Peptides and combination of peptides for use in immunotherapy against various cancers

ABSTRACT

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/266,233, filed 11 Dec. 2015, and Great Britain Application No.1521894.4, filed 11 Dec. 2015, the content of each of these applicationsis herein incorporated by reference in their entirety. This applicationis related to PCT/EP2016/079737, filed 5 Dec. 2016, the content of whichis incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.TXT)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “Sequence_Listing_2912919-059005_ST25.txt” createdon Dec. 8, 2016, and 49,109 bytes in size) is submitted concurrentlywith the instant application, and the entire contents of the SequenceListing are incorporated herein by reference.

FIELD

The present invention relates to peptides, proteins, nucleic acids andcells for use in immunotherapeutic methods. In particular, the presentinvention relates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated T-cell peptide epitopes, aloneor in combination with other tumor-associated peptides that can forexample serve as active pharmaceutical ingredients of vaccinecompositions that stimulate anti-tumor immune responses, or to stimulateT cells ex vivo and transfer into patients. Peptides bound to moleculesof the major histocompatibility complex (MHC), or peptides as such, canalso be targets of antibodies, soluble T-cell receptors, and otherbinding molecules.

The present invention relates to several novel peptide sequences andtheir variants derived from HLA class I molecules of human tumor cellsthat can be used in vaccine compositions for eliciting anti-tumor immuneresponses, or as targets for the development ofpharmaceutically/immunologically active compounds and cells.

BACKGROUND OF THE INVENTION

According to the World Health Organization (WHO), cancer ranged amongthe four major non-communicable deadly diseases worldwide in 2012. Forthe same year, colorectal cancer, breast cancer and respiratory tractcancers were listed within the top 10 causes of death in high incomecountries (http://www.who.int/mediacentre/factsheets/fs310/en/).

Epidemiology

In 2012, 14.1 million new cancer cases, 32.6 million patients sufferingfrom cancer (within 5 years of diagnosis) and 8.2 million cancer deathswere estimated worldwide (Ferlay et al., 2013; Bray et al., 2013). Table1 and Table 2 provide an overview of the estimated incidence, 5 yearprevalence and mortality in different cancer types relevant for thepresent intervention, worldwide and in selected regions, respectively.

TABLE 1 Estimated incidence, 5 year prevalence and mortality ofdifferent cancer types (adult population, both sexes) worldwide in 2012(Ferlay et al., 2013; Bray et al., 2013). Prevalence Cancer Incidence (5year) Mortality Brain, nervous system 256213 342914 189382 Breast1671149 6232108 521907 Colorectum 1360602 3543582 693933 Esophagus455784 464063 400169 Kidney 337860 906746 143406 Leukemia 351965 500934265471 Liver 782451 633170 745533 Lung 1824701 1893078 1589925 Melanoma232130 869754 55488 Ovary 238719 586624 151917 Pancreas 337872 211544330391 Prostate 1094916 3857500 307481 Stomach 951594 1538127 723073Gallbladder 178101 205646 142823 Bladder 429793 1319749 165084 Corpusuteri 319605 1216504 76160 Non-Hodgkin lymphoma 385741 832843 199670

TABLE 2 Estimated incidence, 5 year prevalence and mortality ofdifferent cancer types (adult population, both sexes) in the USA, EU-28,China and Japan in 2012 (Ferlay et al., 2013; Bray et al., 2013).Prevalence Cancer Incidence (5 year) Mortality Brain, nervous system135884 172497 100865 Breast 837245 3358034 197279 Colorectum 8457972334303 396066 Esophagus 294734 323723 255752 Kidney 226733 631350 83741Leukemia 178296 309154 129500 Liver 513172 441007 488485 Lung 12745681394735 1107546 Melanoma 163043 636364 32999 Ovary 108947 270204 65130Pancreas 220842 134864 214886 Prostate 681069 2586710 136419 Stomach615641 1076332 447735 Gallbladder 106202 118588 81391 Bladder 270335879140 91553 Corpus uteri 199211 765101 41734 Non-Hodgkin lymphoma205955 515154 90092

Within the groups of brain cancer, leukemia and lung cancer the currentinvention specifically focuses on glioblastoma (GB), chronic lymphocyticleukemia (CLL) and acute myeloid leukemia (AML), non-small cell andsmall cell lung cancer (NSCLC and SCLC), respectively.

GB is the most common central nervous system malignancy with anage-adjusted incidence rate of 3.19 per 100,000 inhabitants within theUnited States. GB has a very poor prognosis with a 1-year survival rateof 35% and a 5-year survival rate lower than 5%. Male gender, older ageand ethnicity appear to be risk factors for GB (Thakkar et al., 2014).

CLL is the most common leukemia in the Western world where it comprisesabout one third of all leukemia. Incidence rates are similar in the USand Europe, and estimated new cases are about 16,000 per year. CLL ismore common in Caucasians than in Africans, rarer in Hispanics andNative Americans and seldom in Asians. In people of Asian origin, CLLincidence rates are 3 fold lower than in Caucasians (Gunawardana et al.,2008). The five-year overall survival for patients with CLL is about79%.

AML is the second most common type of leukemia diagnosed in both adultsand children. Estimated new cases in the United States are about 21,000per year. The five-year survival rate of people with AML isapproximately 25%.

Lung cancer is the most common type of cancer worldwide and the leadingcause of death from cancer in many countries. Lung cancer is subdividedinto small cell lung cancer and non-small cell lung cancer. NSCLCincludes the histological types adenocarcinoma, squamous cell carcinomaand large cell carcinoma and accounts for 85% of all lung cancers in theUnited States. The incidence of NSCLC is closely correlated with smokingprevalence, including current and former smokers and the five yearsurvival rate was reported to be 15% (World Cancer Report, 2014; Molinaet al., 2008).

Therapy

Breast Cancer

Breast cancer is an immunogenic cancer entity and different types ofinfiltrating immune cells in primary tumors exhibit distinct prognosticand predictive significance. A large number of early phase immunotherapytrials have been conducted in breast cancer patients. Most of thecompleted vaccination studies targeted HER2 and carbohydrate antigenslike MUC-1 and revealed rather disappointing results. Clinical data onthe effects of immune checkpoint modulation with ipilimumab and other Tcell-activating antibodies in breast cancer patients are emerging(Emens, 2012).

Chronic Lymphocytic Leukemia

While CLL is not curable at present, many patients show only slowprogression of the disease or worsening of symptoms. As patients do notbenefit from an early onset of treatment, the initial approach is “watchand wait” (Richards et al., 1999). For patients with symptomatic orrapidly progressing disease, several treatment options are available.These include chemotherapy, targeted therapy, immune-based therapieslike monoclonal antibodies, chimeric antigen-receptors (CARs) and activeimmunotherapy, and stem cell transplants.

Monoclonal antibodies are widely used in hematologic malignancies. Thisis due to the knowledge of suitable antigens based on the goodcharacterization of immune cell surface molecules and the accessibilityof tumor cells in blood or bone marrow. Common monoclonal antibodiesused in CLL therapy target either CD20 or CD52. Rituximab, the firstmonoclonal anti-CD20 antibody originally approved by the FDA fortreatment of NHLs, is now widely used in CLL therapy. Combinationaltreatment with rituximab/fludarabine/cyclophosphamide leads to higher CRrates and improved overall survival (OS) compared to the combinationfludarabine/cyclophosphamide and has become the preferred treatmentoption (Hallek et al., 2008). Ofatumomab targets CD20 and is used fortherapy of refractory CLL patients (Wierda et al., 2011). Obinutuzumabis another monoclonal anti-CD20 antibody used in first-line treatment incombination with chlorambucil (Goede et al., 2014).

Alemtuzumab is an anti-CD52 antibody used for treatment of patients withchemotherapy-resistant disease or patients with poor prognostic factorsas del 17p or p53 mutations (Parikh et al., 2011). Novel monoclonalantibodies target CD37 (otlertuzumab, BI 836826, IMGN529 and(177)Lu-tetulomab) or CD40 (dacetuzumab and lucatumumab) and are testedin pre-clinical settings (Robak and Robak, 2014).

Several completed and ongoing trials are based on engineered autologouschimeric antigen receptor (CAR)-modified T cells with CD19 specificity(Maus et al., 2014). So far, only the minority of patients showeddetectable or persistent CARs. One partial response (PR) and twocomplete responses (CR) have been detected in the CAR T-cell trials byPorter et al. and Kalos et al. (Kalos et al., 2011; Porter et al.,2011).

Active immunotherapy includes the following strategies: gene therapy,whole modified tumor cell vaccines, DC-based vaccines and tumorassociated antigen (TAA)-derived peptide vaccines.

Approaches in gene therapy make use of autologous genetically modifiedtumor cells. These B-CLL cells are transfected withimmuno-(co-)stimulatory genes like IL-2, IL-12, TNF-alpha, GM-CSF, CD80,CD40L, LFA-3 and ICAM-1 to improve antigen presentation and T cellactivation (Carballido et al., 2012). While specific T-cell responsesand reduction in tumor cells are readily observed, immune responses areonly transient.

Several studies have used autologous DCs as antigen presenting cells toelicit anti-tumor responses. DCs have been loaded ex vivo with tumorassociated peptides, whole tumor cell lysate and tumor-derived RNA orDNA. Another strategy uses whole tumor cells for fusion with DCs andgeneration of DC-B-CLL-cell hybrids. Transfected DCs initiated both CD4+and CD8+ T-cell responses (Muller et al., 2004). Fusion hybrids and DCsloaded with tumor cell lysate or apoptotic bodies increasedtumor-specific CD8+ T-cell responses. Patients that showed a clinicalresponse had increased IL-12 serum levels and reduced numbers of Tregs(Palma et al., 2008).

Different approaches use altered tumor cells to initiate or increaseCLL-specific immune responses. An example for this strategy is thegeneration of trioma cells: B-CLL cells are fused to anti-Fc receptorexpressing hybridoma cells that have anti-APC specificity. Trioma cellsinduced CLL-specific T-cell responses in vitro (Kronenberger et al.,2008).

Another strategy makes use of irradiated autologous CLL cells withBacillus Calmette-Guérin as an adjuvant as a vaccine. Several patientsshowed a reduction in leukocyte levels or stable disease (Hus et al.,2008).

Besides isolated CLL cells, whole blood from CLL patients has been usedas a vaccine after preparation in a blood treatment unit. The vaccineelicited CLL-specific T-cell responses and led to partial clinicalresponses or stable disease in several patients (Spaner et al., 2005).

Several TAAs are over-expressed in CLL and are suitable forvaccinations. These include fibromodulin (Mayr et al., 2005),RHAMM/CD168 (Giannopoulos et al., 2006), MDM2 (Mayr et al., 2006), hTERT(Counter et al., 1995), the oncofetal antigen-immature laminin receptorprotein (OFAiLRP) (Siegel et al., 2003), adipophilin (Schmidt et al.,2004), survivin (Granziero et al., 2001), KW1 to KW14 (Krackhardt etal., 2002) and the tumor-derived IgVHCDR3 region (Harig et al., 2001;Carballido et al., 2012). A phase I clinical trial was conducted usingthe RHAMM-derived R3 peptide as a vaccine. 5 of 6 patients haddetectable R3-specific CD8+ T-cell responses (Giannopoulos et al.,2010).

Colorectal Cancer

Depending on the colorectal cancer (CRC) stage, different standardtherapies are available for colon and rectal cancer. Standard proceduresinclude surgery, radiation therapy, chemotherapy and targeted therapyfor CRC (Berman et al., 2015a; Berman et al., 2015b).

Removal of the tumor is essential for the treatment of CRC. Forchemotherapeutic treatment, the drugs capecitabine or 5-fluorouracil(5-FU) are used. For combinational chemotherapy, a cocktail containing5-FU, leucovorin and oxaliplatin (FOLFOX) is recommended (Stintzing,2014; Berman et al., 2015b). In addition to chemotherapeutic drugs,several monoclonal antibodies targeting the epidermal growth factorreceptor (EGFR, cetuximab, panitumumab) or the vascular endothelialgrowth factor-A (VEGF-A, bevacizumab) are administered to patients withhigh stage disease. For second-line and later treatment the inhibitorfor VEGF aflibercept, the tyrosine kinase inhibitor regorafenib and thethymidylate-synthetase inhibitor TAS-102 and the dUTPase inhibitorTAS-114 can be used (Stintzing, 2014; Wilson et al., 2014).

Latest clinical trials analyze active immunotherapy as a treatmentoption against CRC. Those strategies include the vaccination withpeptides from tumor-associated antigens (TAAs), whole tumor cells,dendritic cell (DC) vaccines and viral vectors (Koido et al., 2013).

Peptide vaccines have so far been directed against carcinoembryonicantigen (CEA), mucin 1, EGFR, squamous cell carcinoma antigen recognizedby T cells 3 (SART3), beta-human chorionic gonadotropin (beta-hCG),Wilms' Tumor antigen 1 (WT1), Survivin-2B, MAGE3, p53, ring fingerprotein 43 and translocase of the outer mitochondrial membrane 34(TOMM34), or mutated KRAS. In several phase I and II clinical trialspatients showed antigen-specific CTL responses or antibody production.In contrast to immunological responses, many patients did not benefitfrom peptide vaccines on the clinical level (Koido et al., 2013; Miyagiet al., 2001; Moulton et al., 2002; Okuno et al., 2011).

Dendritic cell vaccines comprise DCs pulsed with either TAA-derivedpeptides, tumor cell lysates, apoptotic tumor cells, or tumor RNA orDC-tumor cell fusion products. While many patients in phase I/II trialsshowed specific immunological responses, only the minority had aclinical benefit (Koido et al., 2013).

Whole tumor cell vaccines consist of autologous tumor cells modified tosecrete GM-CSF, modified by irradiation or virus-infected, irradiatedcells. Most patients showed no clinical benefit in several phase II/IIItrials (Koido et al., 2013).

Vaccinia virus or replication-defective avian poxvirus encoding CEA aswell as B7.1, ICAM-1 and LFA-3 have been used as vehicles in viralvector vaccines in phase I clinical trials. A different study usednon-replicating canary pox virus encoding CEA and B7.1. Besides theinduction of CEA-specific T cell responses 40% of patients showedobjective clinical responses (Horig et al., 2000; Kaufman et al., 2008).

Esophageal Cancer

Immunotherapy may be a promising novel approach to treat advancedesophageal cancer. Several cancer-associated genes and cancer-testisantigens were shown to be over-expressed in esophageal cancer, includingdifferent MAGE genes, NY-ESO-1 and EpCAM (Kimura et al., 2007; Liang etal., 2005; Inoue et al., 1995; Bujas et al., 2011; Tanaka et al., 1997;Quillien et al., 1997). Those genes represent very interesting targetsfor immunotherapy and most of them are under investigation for thetreatment of other malignancies (ClinicalTrials.gov, 2015). Furthermore,up-regulation of PD-L1 and PD-L2 was described in esophageal cancer,which correlated with poorer prognosis. Thus, esophageal cancer patientswith PD-L1-positive tumors might benefit from anti-PD-L1 immunotherapy(Ohigashi et al., 2005).

Clinical data on immunotherapeutic approaches in esophageal cancer arestill relatively scarce at present, as only a very limited number ofearly phase clinical trials have been completed. A vaccine consisting ofthree peptides derived from three different cancer-testis antigens (TTKprotein kinase, lymphocyte antigen 6 complex locus K and insulin-likegrowth factor (IGF)-II mRNA binding protein 3) was administered topatients with advanced esophageal cancer in a phase I trial withmoderate results. Intra-tumoral injection of activated T cells after invitro challenge with autologous malignant cells elicited complete orpartial tumor responses in four of eleven patients in a phase I/II study(Toomey et al., 2013). A vaccine consisting of three peptides derivedfrom three different cancer-testis antigens (TTK protein kinase,lymphocyte antigen 6 complex locus K and insulin-like growth factor(IGF)-II mRNA binding protein 3) was administered to patients withadvanced esophageal cancer in a phase I trial with moderate results(Kono et al., 2009). Intra-tumoral injection of activated T cells afterin vitro challenge with autologous malignant cells and interleukin 2elicited complete or partial tumor responses in four of eleven patientsin a phase I/II study (Toh et al., 2000; Toh et al., 2002). Furtherclinical trials are currently performed to evaluate the impact ofdifferent immunotherapies on esophageal cancer, including adoptivecellular therapy (NCT01691625, NCT01691664, NCT01795976, NCT02096614,NCT02457650) vaccination strategies (NCT01143545, NCT01522820) andanti-PD-L1 therapy (NCT02340975) (ClinicalTrials.gov, 2015).

Gastric Cancer

The efficacy of current therapeutic regimens for advanced GC is poor,resulting in low 5-year survival rates. Immunotherapy might be analternative approach to ameliorate the survival of GC patients. Adoptivetransfer of tumor-associated lymphocytes and cytokine induced killercells, peptide-based vaccines targeting HER2/neu, MAGE-3 or vascularendothelial growth factor receptor 1 and 2 and dendritic cell-basedvaccines targeting HER2/neu showed promising results in clinical GCtrials. Immune checkpoint inhibition and engineered T cells mightrepresent additional therapeutic options, which is currently evaluatedin pre-clinical and clinical studies (Matsueda and Graham, 2014).

Glioblastoma

The therapeutic options for glioblastoma (WHO grade IV) are verylimited. Different immunotherapeutic approaches are investigated for thetreatment of GB, including immune-checkpoint inhibition, vaccination andadoptive transfer of engineered T cells.

Antibodies directed against inhibitory T cell receptors or their ligandswere shown to efficiently enhance T cell-mediated anti-tumor immuneresponses in different cancer types, including melanoma and bladdercancer. The effects of T cell activating antibodies like ipilimumab andnivolumab are therefore assessed in clinical GB trials, but preliminarydata indicate autoimmune-related adverse events.

Different vaccination strategies for GB patients are currentlyinvestigated, including peptide-based vaccines, heat-shock proteinvaccines, autologous tumor cell vaccines, dendritic cell-based vaccinesand viral protein-based vaccines. In these approaches peptides derivedfrom GB-associated proteins like epidermal growth factor receptorvariant III (EG-FRvIII) or heat shock proteins or dendritic cells pulsedwith autologous tumor cell lysate or cytomegalo virus components areapplied to induce an anti-tumor immune response in GB patients. Severalof these studies reveal good safety and tolerability profiles as well aspromising efficacy data.

Adoptive transfer of genetically modified T cells is an additionalimmunotherapeutic approach for the treatment of GB. Different clinicaltrials currently evaluate the safety and efficacy of chimeric antigenreceptor bearing T cells directed against HER2, IL-13 receptor alpha 2and EGFRvIII (Ampie et al., 2015).

Liver Cancer

Therapeutic options in advanced non-resectable HCC are limited toSorafenib, a multi-tyrosine kinase inhibitor (Chang et al., 2007;Wilhelm et al., 2004). Sorafenib is the only systemic drug confirmed toincrease survival by about 3 months and currently represents the onlyexperimental treatment option for such patients (Chapiro et al., 2014;Llovet et al., 2008). Lately, a limited number of immunotherapy trialsfor HCC have been conducted. Cytokines have been used to activatesubsets of immune cells and/or increase the tumor immunogenicity(Reinisch et al., 2002; Sangro et al., 2004). Other trials have focusedon the infusion of Tumor-infiltrating lymphocytes or activatedperipheral blood lymphocytes (Shi et al., 2004; Takayama et al., 1991;Takayama et al., 2000).

So far, a small number of therapeutic vaccination trials have beenexecuted. Butterfield et al. conducted two trials using peptides derivedfrom alpha-fetoprotein (AFP) as a vaccine or DCs loaded with AFPpeptides ex vivo (Butterfield et al., 2003; Butterfield et al., 2006).In two different studies, autologous dendritic cells (DCs) were pulsedex vivo with autologous tumor lysate (Lee et al., 2005) or lysate of thehepatoblastoma cell line HepG2 (Palmer et al., 2009). So far,vaccination trials have only shown limited improvements in clinicaloutcomes.

Melanoma

Enhancing the anti-tumor immune responses appears to be a promisingstrategy for the treatment of advanced melanoma. In the United Statesthe immune checkpoint inhibitor ipilimumab as well as the BRAF kinaseinhibitors vemurafenib and dabrafenib and the MEK inhibitor trametinibare already approved for the treatment of advanced melanoma. Bothapproaches increase the patient's anti-tumor immunity—ipilimumabdirectly by reducing T cell inhibition and the kinase inhibitorsindirectly by enhancing the expression of melanocyte differentiationantigens. Additional checkpoint inhibitors (nivolumab and lambrolizumab)are currently investigated in clinical studies with first encouragingresults. Additionaly, different combination therapies targeting theanti-tumor immune response are tested in clinical trials includingipilimumab plus nivolumab, ipilimumab plus a gp100-derived peptidevaccine, ipilimumab plus dacarbazine, ipilimumab plus IL-2 and iplimumabplus GM-CSF (Srivastava and McDermott, 2014).

Several different vaccination approaches have already been evaluated inpatients with advanced melanoma. So far, phase III trials revealedrather disappointing results and vaccination strategies clearly need tobe improved. Therefore, new clinical trials, like the OncoVEX GM-CSFtrial or the DERMA trial, aim at improving clinical efficacy withoutreducing tolerability (http://www.cancerresearchuk.org).

Adoptive T cell transfer shows great promise for the treatment ofadvanced stage melanoma. In vitro expanded autologous tumor infiltratinglymphocytes as well as T cells harboring a high affinity T cell receptorfor the cancer-testis antigen NY-ESO-1 had significant beneficial andlow toxic effects upon transfer into melanoma patients. Unfortunately, Tcells with high affinity T cell receptors for the melanocyte specificantigens MART1 and gp100 and the cancer-testis antigen MAGEA3 inducedconsiderable toxic effects in clinical trials. Thus, adoptive T celltransfer has high therapeutic potential, but safety and tolerability ofthese treatments needs to be further increased (Phan and Rosenberg,2013; Hinrichs and Restifo, 2013).

Non-Small Cell Lung Cancer

Because the disease has usually spread by the time it is discovered,radiation therapy and chemotherapy are often used, sometimes incombination with surgery (S3-Leitlinie Lungenkarzinom, 2011). To expandthe therapeutic options for NSCLC, different immunotherapeuticapproaches have been studied or are still under investigation. Whilevaccination with L-BLP25 or MAGEA3 failed to demonstrate avaccine-mediated survival advantage in NSCLC patients, an allogeneiccell line-derived vaccine showed promising results in clinical studies.Additionally, further vaccination trials targeting gangliosides, theepidermal growth factor receptor and several other antigens arecurrently ongoing. An alternative strategy to enhance the patient'santi-tumor T cell response consists of blocking inhibitory T cellreceptors or their ligands with specific antibodies. The therapeuticpotential of several of these antibodies, including ipilimumab,nivolumab, pembrolizumab, MPDL3280A and MEDI-4736, in NSCLC is currentlyevaluated in clinical trials (Reinmuth et al., 2015).

Ovarian Cancer

Immunotherapy appears to be a promising strategy to ameliorate thetreatment of ovarian cancer patients, as the presence ofpro-inflammatory tumor infiltrating lymphocytes, especially CD8-positiveT cells, correlates with good prognosis and T cells specific fortumor-associated antigens can be isolated from cancer tissue.

Therefore, a lot of scientific effort is put into the investigation ofdifferent immunotherapies in ovarian cancer. A considerable number ofpre-clinical and clinical studies have already been performed andfurther studies are currently ongoing. Clinical data are available forcytokine therapy, vaccination, monoclonal antibody treatment, adoptivecell transfer and immunomodulation.

Cytokine therapy with interleukin-2, interferon-alpha, interferon-gammaor granulocyte-macrophage colony stimulating factor aims at boosting thepatient's own anti-tumor immune response and these treatments havealready shown promising results in small study cohorts.

Phase I and II vaccination studies, using single or multiple peptides,derived from several tumor-associated proteins (Her2/neu, NY-ESO-1, p53,Wilms tumor-1) or whole tumor antigens, derived from autologous tumorcells revealed good safety and tolerability profiles, but only low tomoderate clinical effects.

Monoclonal antibodies that specifically recognize tumor-associatedproteins are thought to enhance immune cell-mediated killing of tumorcells. The anti-CA-125 antibodies oregovomab and abagovomab as well asthe anti-EpCAM antibody catumaxomab achieved promising results in phaseII and III studies. In contrast, the anti-MUC1 antibody HMFG1 failed toclearly enhance survival in a phase III study.

An alternative approach uses monoclonal antibodies to target and blockgrowth factor and survival receptors on tumor cells. Whileadministration of trastuzumab (anti-HER2/neu antibody) and MOv18 andMORAb-003 (anti-folate receptor alpha antibodies) only conferred limitedclinical benefit to ovarian cancer patients, addition of bevacizumab(anti-VEGF antibody) to the standard chemotherapy in advanced ovariancancer appears to be advantageous.

Adoptive transfer of immune cells achieved heterogeneous results inclinical trials. Adoptive transfer of autologous, in vitro expandedtumor infiltrating T cells was shown to be a promising approach in apilot trial. In contrast, transfer of T cells harboring a chimericantigen receptor specific for folate receptor alpha did not induce asignificant clinical response in a phase I trial. Dendritic cells pulsedwith tumor cell lysate or tumor-associated proteins in vitro were shownto enhance the anti-tumor T cell response upon transfer, but the extentof T cell activation did not correlate with clinical effects. Transferof natural killer cells caused significant toxicities in a phase IIstudy.

Intrinsic anti-tumor immunity as well as immunotherapy are hampered byan immunosuppressive tumor microenvironment. To overcome this obstacleimmunomodulatory drugs, like cyclophosphamide, anti-CD25 antibodies andpegylated liposomal doxorubicin are tested in combination withimmunotherapy. Most reliable data are currently available foripilimumab, an anti-CTLA4 antibody, which enhances T cell activity.Ipilimumab was shown to exert significant anti-tumor effects in ovariancancer patients (Mantia-Smaldone et al., 2012).

Pancreatic Cancer

Therapeutic options for pancreatic cancer patients are very limited. Onemajor problem for effective treatment is the typically advanced tumorstage at diagnosis. Vaccination strategies are investigated as furtherinnovative and promising alternative for the treatment of pancreaticcancer. Peptide-based vaccines targeting KRAS mutations, reactivetelomerase, gastrin, survivin, CEA and MUC1 have already been evaluatedin clinical trials, partially with promising results. Furthermore,clinical trials for dendritic cell-based vaccines, allogeneicGM-CSF-secreting vaccines and algenpantucel-L in pancreatic cancerpatients also revealed beneficial effects of immunotherapy. Additionalclinical trials further investigating the efficiency of differentvaccination protocols are currently ongoing (Salman et al., 2013).

Prostate Cancer

The dendritic cell-based vaccine sipuleucel-T was the first anti-cancervaccine to be approved by the FDA. Due to its positive effect onsurvival in patients with CRPC, much effort is put into the developmentof further immunotherapies. Regarding vaccination strategies, thepeptide vaccine prostate-specific antigen (PSA)-TRICOM, the personalizedpeptide vaccine PPV, the DNA vaccine pTVG-HP and the whole cell vaccineexpressing GM-CSF GVAX showed promising results in different clinicaltrials. Furthermore, dendritic cell-based vaccines other thansipuleucel-T, namely BPX-101 and DCVAC/Pa were shown to elicitedclinical responses in prostate cancer patients. Immune checkpointinhibitors like ipilimumab and nivolumab are currently evaluated inclinical studies as monotherapy as well as in combination with othertreatments, including androgen deprivation therapy, local radiationtherapy, PSA-TRICOM and GVAX. The immunomodulatory substancetasquinimod, which significantly slowed progression and increasedprogression free survival in a phase II trial, is currently furtherinvestigated in a phase III trial. Lenalidomide, anotherimmunomodulator, induced promising effects in early phase clinicalstudies, but failed to improve survival in a phase III trial. Despitethese disappointing results further lenalidomide trials are ongoing(Quinn et al., 2015).

Renal Cell Carcinoma

The known immunogenity of RCC has represented the basis supporting theuse of immunotherapy and cancer vaccines in advanced RCC. Theinteresting correlation between lymphocytes PD-1 expression and RCCadvanced stage, grade and prognosis, as well as the selective PD-L1expression by RCC tumor cells and its potential association with worseclinical outcomes, have led to the development of new anti PD-1/PD-L1agents, alone or in combination with anti-angiogenic drugs or otherimmunotherapeutic approaches, for the treatment of RCC (Massari et al.,2015). In advanced RCC, a phase III cancer vaccine trial called TRISTstudy evaluates whether TroVax (a vaccine using a tumor-associatedantigen 5T4, with a pox virus vector), added to first-line standard ofcare therapy, prolongs survival of patients with locally advanced ormRCC. Median survival had not been reached in either group with 399patients (54%) remaining on study however analysis of the data confirmsprior clinical results, demonstrating that TroVax is bothimmunologically active and that there is a correlation between thestrength of the 5T4-specific antibody response and improved survival.Further there are several studies searching for peptide vaccines usingepitopes being over-expressed in RCC.

Various approaches of tumor vaccines have been under investigation.Studies using whole-tumor approaches, including tumor cell lysates,fusions of dendritic cells with tumor cells, or whole-tumor RNA weredone in RCC patients, and remissions of tumor lesions were reported insome of these trials (Avigan et al., 2004; Holtl et al., 2002; Marten etal., 2002; Su et al., 2003; Wittig et al., 2001).

Small Cell Lung Cancer

Innovations occurred regarding detection, diagnosis and treatment ofSCLC. It was shown that the usage of CT scans instead of x-rays forearly cancer detection lowered the risk of death from lung cancer.Nowadays, the diagnosis of SCLC can be supported by fluorescence orvirtual bronchoscopy; the real-time tumor imagining can be implementedby the radiation treatment. The novel anti-angiogenesis drugs likebevacizumab (Avastin), sunitinib (Sutent) and nintedanib (BIBF 1120)were shown to have therapeutically effects in treatment of SCLC(American Cancer Society, 2015). The immune therapy presents anexcessively investigated field of cancer therapy. Various approaches arestudded in the treatment of SCLC. One of the approaches targets theblocking of CTLA-4, a natural human immune suppressor. The inhibition ofCTLA-4 intends to boost the immune system to combat the cancer.Recently, the development of promising immune check point inhibitors fortreatment of SCLC has been started. Another approach is based onanti-cancer vaccines which is currently available for treatment of SCLCin clinical studies (American Cancer Society, 2015; National CancerInstitute (NCI), 2011).

Acute Myeloid Leukemia

One treatment option is targeting CD33 with antibody-drug conjugates(anti-CD33+ calechiamicin, SGN-CD33a, anti-CD33+actinium-225),bispecific antibodies (recognition of CD33+CD3 (AMG 330) or CD33+CD16)and chimeric antigen receptors (CARs) (Estey, 2014).

Non-Hodgkin Lymphoma

Treatment of NHL depends on the histologic type and stage (NationalCancer Institute, 2015). Spontaneous tumor regression can be observed inlymphoma patients. Therefore, active immunotherapy is a therapy option(Palomba, 2012). An important vaccination option includes Id vaccines. Blymphocytes express surface immunoglobulins with a specific amino acidsequence in the variable regions of their heavy and light chains, uniqueto each cell clone (=idiotype, Id). The idiotype functions as a tumorassociated antigen. Passive immunization includes the injection ofrecombinant murine anti-Id monoclonal antibodies alone or in combinationwith IFNalpha, IL2 or chlorambucil.

Active immunization includes the injection of recombinant protein (Id)conjugated to an adjuvant (KLH), given together with GM-CSF as an immuneadjuvant. Tumor-specific Id is produced by hybridoma cultures or usingrecombinant DNA technology (plasmids) by bacterial, insect or mammaliancell culture. Three phase III clinical trials have been conducted(Biovest, Genitope, Favrille). In two trials patients had receivedrituximab. GM-CSF was administered in all three trials. Biovest usedhybridoma-produced protein, Genitope and Favrille used recombinantprotein. In all three trials Id was conjugated to KLH. Only Biovest hada significant result.

Vaccines other than Id include the cancer-testis antigens MAGE, NY-ESO1and PASD-1, the B-cell antigen CD20 or cellular vaccines. The latestmentioned consist of DCs pulsed with apoptotic tumor cells, tumor celllysate, DC-tumor cell fusion or DCs pulsed with tumor-derived RNA. Insitu vaccination involves the vaccination with intra-tumoral CpG incombination with chemotherapy or irradiated tumor cells grown in thepresence of GM-CSF and collection/expansion/re-infusion of T cells.Vaccination with antibodies that alter immunologic checkpoints arecomprised of anti-CD40, anti-OX40, anti-41 BB, anti-CD27, anti-GITR(agonist antibodies that directly enhance anti-tumor response) oranti-PD1, anti-CTLA-4 (blocking antibodies that inhibit the checkpointthat would hinder the immune response). Examples are ipilimumab(anti-CTLA-4) and CT-011 (anti-PD1) (Palomba, 2012).

Uterine Cancer

There are also some immunotherapeutic approaches that are currentlybeing tested. In a Phase I/II Clinical Trial patients suffering fromuterine cancer were vaccinated with autologous dendritic cells (DCs)electroporated with Wilms' tumor gene 1 (WT1) mRNA. Besides one case oflocal allergic reaction to the adjuvant, no adverse side effects wereobserved and 3 out of 6 patients showed an immunological response(Coosemans et al., 2013).

As stated above, HPV infections provoke lesions that may ultimately leadto cervical cancer. Therefore, the HPV viral oncoproteins E6 and E7 thatare constitutively expressed in high-grade lesions and cancer and arerequired for the onset and maintenance of the malignant phenotype areconsidered promising targets for immunotherapeutic approaches (Hung etal., 2008; Vici et al., 2014). One study performed Adoptive T-celltherapy (ACT) in patients with metastatic cervical cancer. Patientsreceive an infusion with E6 and E7 reactive tumor-infiltrating T cells(TILs) resulting in complete regression in 2 and a partial response in 1out of 9 patients (Stevanovic et al., 2015). Furthermore, anintracellular antibody targeting E7 was reported to block tumor growthin mice (Accardi et al., 2014). Also peptide, DNA and DC-based vaccinestargeting HPV E6 and E7 are in clinical trials (Vici et al., 2014).

Gallbladder Adenocarcinoma and Cholangiocarcinoma

Cholangiocarcinoma (CCC) is mostly identified in advanced stages becauseit is difficult to diagnose. Gallbladder cancer (GBC) is the most commonand aggressive malignancy of the biliary tract worldwide. As for GBConly 10% of tumors are resectable and even with surgery most progress tometastatic disease, prognosis is even worse than for CCC with a 5-yearsurvival of less than 5%. Although most tumors are unresectable there isstill no effective adjuvant therapy (Rakic et al., 2014). Some studiesshowed that combination of chemotherapeutic drugs or combination oftargeted therapy (anti-VEGFR/EGFR) with chemotherapy led to an increasedoverall survival and might be promising treatment options for the future(Kanthan et al., 2015). Due to the rarity of carcinomas of the biliarytract in general there are only a few GBC or CCC specific studies, whilemost of them include all biliary tract cancers. This is the reason whytreatment did not improve during the last decades and RO resection stillis the only curative treatment option.

Urinary Bladder Cancer

The standard treatment for bladder cancer includes surgery, radiationtherapy, chemotherapy and immunotherapy.

An effective immunotherapeutic approach is established in the treatmentof aggressive non-muscle invasive bladder cancer (NMIBC). Thereby, aweakened form of the bacterium Mycobacterium bovis (bacillusCalmette-Guérin=BCG) is applied as an intravesical solution. The majoreffect of BCG treatment is a significant long-term (up to 10 years)protection from cancer recurrence and reduced progression rate. Inprinciple, the treatment with BCG induces a local inflammatory responsewhich stimulates the cellular immune response. The immune response toBCG is based on the following key steps: infection of urothelial andbladder cancer cells by BCG, followed by increased expression ofantigen-presenting molecules, induction of immune response mediated viacytokine release, induction of antitumor activity via involvement ofvarious immune cells (thereunder cytotoxic T lymphocytes, neutrophils,natural killer cells, and macrophages) (Fuge et al., 2015; Gandhi etal., 2013).

BCG treatment is in general well tolerated by patients but can be fatalespecially by the immunocompromised patients. BCG refractory is observedin about 30-40% of patients (Fuge et al., 2015; Steinberg et al.,2016a). The treatment of patients who failed the BCG therapy ischallenging. The patients who failed the BCG treatment are at high riskfor developing of muscle-invasive disease. Radical cystectomy is thepreferable treatment option for non-responders (Steinberg et al., 2016b;von Rundstedt and Lerner, 2015). The FDA approved second line therapy ofBCG-failed NMIBC for patients who desire the bladder preservation is thechemotherapeutic treatment with valrubicin. A number of other secondline therapies are available or being currently under investigation aswell, thereunder immunotherapeutic approaches like combinedBCG-interferon or BCG-check point inhibitor treatments, pre-BCGtransdermal vaccination, treatment with Mycobacterium phlei cellwall-nucleic acid (MCNA) complex, mono- or combination chemotherapy withvarious agents like mitomycin C, gemcitabine, docetaxel, nab-paclitaxel,epirubicin, mitomycin/gemcitabine, gemcitabine/docetaxel, anddevice-assisted chemotherapies like thermochemo-, radiochemo-,electromotive or photodynamic therapies (Fuge et al., 2015; Steinberg etal., 2016b; von Rundstedt and Lerner, 2015). Further evaluation ofavailable therapies in clinical trials is still required.

The alternative treatment options for advanced bladder cancer are beinginvestigated in ongoing clinical trials. The current clinical trialsfocused on the development of molecularly targeted therapies andimmunotherapies. The targeted therapies investigate the effects ofcancerogenesis related pathway inhibitors (i.e. mTOR, vascularendothelial, fibroblast, or epidermal growth factor receptors,anti-angiogenesis or cell cycle inhibitors) in the treatment of bladdercancer. The development of molecularly targeted therapies remainschallenging due to high degree of genetic diversity of bladder cancer.The main focus of the current immunotherapy is the development ofcheckpoint blockage agents like anti-PD1 monoclonal antibody andadoptive T-cell transfer (Knollman et al., 2015; Grivas et al., 2015;Jones et al., 2016; Rouanne et al., 2016).

Head and Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinomas (HNSCC) are heterogeneous tumorswith differences in epidemiology, etiology and treatment (Economopoulouet al., 2016). Treatment for early HNSCC involves single-modalitytherapy with either surgery or radiation (World Health Organization,2014). Advanced cancers are treated by a combination of chemotherapywith surgery and/or radiation therapy.

HNSCC is considered an immunosuppressive disease, characterized by thedysregulation of immunocompetent cells and impaired cytokine secretion(Economopoulou et al., 2016). Immunotherapeutic strategies differbetween HPV-negative and HPV-positive tumors.

In HPV-positive tumors, the viral oncoproteins E6 and E7 represent goodtargets, as they are continuously expressed by tumor cells and areessential to maintain the transformation status of HPV-positive cancercells. Several vaccination therapies are currently under investigationin HPV-positive HNSCC, including DNA vaccines, peptide vaccines andvaccines involving dendritic cells (DCs). Additionally, an ongoing phaseII clinical trial investigates the efficacy of lymphodepletion followedby autologous infusion of TILs in patients with HPV-positive tumors(Economopoulou et al., 2016).

In HPV-negative tumors, several immunotherapeutic strategies arecurrently used and under investigation. The chimeric IgG1 anti-EGFRmonoclonal antibody cetuximab has been approved by the FDA incombination with chemotherapy as standard first line treatment forrecurring/metastatic HNSCC. Other anti-EGFR monoclonal antibodies,including panitumumab, nimotuzumab and zalutumumab, are evaluated inHNSCC. Several immune checkpoint inhibitors are investigated in clinicaltrials for their use in HNSCC. They include the following antibodies:Ipilimumab (anti-CTLA-4), tremelimumab (anti-CTLA-4), pembrolizumab(anti-PD-1), nivolumab (anti-PD-1), durvalumab (anti-PD-1), anti-KIR,urelumab (anti-CD137), and anti-LAG-3.

Two clinical studies with HNSCC patients evaluated the use of DCs loadedwith p53 peptides or apoptotic tumor cells. The immunological responseswere satisfactory and side effects were acceptable. Several studies havebeen conducted using adoptive T cell therapy (ACT). T cells were inducedagainst either irradiated autologous tumor cells or EBV. Results indisease control and overall survival were promising (Economopoulou etal., 2016).

Considering the severe side-effects and expense associated with treatingcancer, there is a need to identify factors that can be used in thetreatment of cancer in general and glioblastoma (GB), breast cancer(BRCA), colorectal cancer (CRC), renal cell carcinoma (RCC), chroniclymphocytic leukemia (CLL), hepatocellular carcinoma (HCC), non-smallcell and small cell lung cancer (NSCLC, SCLC), Non-Hodgkin lymphoma(NHL), acute myeloid leukemia (AML), ovarian cancer (OC), pancreaticcancer (PC), prostate cancer (PCA), esophageal cancer including cancerof the gastric-esophageal junction (OSCAR), gallbladder cancer andcholangiocarcinoma (GBC, CCC), melanoma (MEL), gastric cancer (GC),urinary bladder cancer (UBC), head and neck squamous cell carcinoma(HNSCC), and uterine cancer (UEC) in particular. There is also a need toidentify factors representing biomarkers for cancer in general and theabove-mentioned cancer types in particular, leading to better diagnosisof cancer, assessment of prognosis, and prediction of treatment success.

Immunotherapy of cancer represents an option of specific targeting ofcancer cells while minimizing side effects. Cancer immunotherapy makesuse of the existence of tumor associated antigens.

The current classification of tumor associated antigens (TAAs) comprisesthe following major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members and NY-ESO-1.

b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose. Most of the knowndifferentiation antigens are found in melanomas and normal melanocytes.Many of these melanocyte lineage-related proteins are involved inbiosynthesis of melanin and are therefore not tumor specific butnevertheless are widely used for cancer immunotherapy. Examples include,but are not limited to, tyrosinase and Melan-A/MART-1 for melanoma orPSA for prostate cancer.

c) Over-expressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir over-expression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, survivin, telomerase, or WT1.

d) Tumor-specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor-specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors. Tumor-specificity (or-association) of a peptide may also arise if the peptide originates froma tumor-(-associated) exon in case of proteins with tumor-specific(-associated) isoforms.

e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.

f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

T-cell based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). The antigensthat are recognized by the tumor specific T lymphocytes, that is, theepitopes thereof, can be molecules derived from all protein classes,such as enzymes, receptors, transcription factors, etc. which areexpressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides.

MHC class I molecules can be found on most nucleated cells. They presentpeptides that result from proteolytic cleavage of predominantlyendogenous proteins, defective ribosomal products (DRIPs) and largerpeptides. However, peptides derived from endosomal compartments orexogenous sources are also frequently found on MHC class I molecules.This non-classical way of class I presentation is referred to ascross-presentation in the literature (Brossart and Bevan, 1997; Rock etal., 1990). MHC class II molecules can be found predominantly onprofessional antigen presenting cells (APCs), and primarily presentpeptides of exogenous or transmembrane proteins that are taken up byAPCs e.g. during endocytosis, and are subsequently processed.

Complexes of peptide and MHC class I are recognized by CD8-positive Tcells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T cells bearing the appropriate TCR. It is wellknown that the TCR, the peptide and the MHC are thereby present in astoichiometric amount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells. Theidentification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) is of great importance for the development ofpharmaceutical products for triggering anti-tumor immune responses(Gnjatic et al., 2003). At the tumor site, T helper cells, support acytotoxic T cell- (CTL-) friendly cytokine milieu (Mortara et al., 2006)and attract effector cells, e.g. CTLs, natural killer (NK) cells,macrophages, and granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules (Dengjel et al.,2006).

Elongated (longer) peptides of the invention can act as MHC class IIactive epitopes.

T-helper cells, activated by MHC class II epitopes, play an importantrole in orchestrating the effector function of CTLs in anti-tumorimmunity. T-helper cell epitopes that trigger a T-helper cell responseof the TH1 type support effector functions of CD8-positive killer Tcells, which include cytotoxic functions directed against tumor cellsdisplaying tumor-associated peptide/MHC complexes on their cellsurfaces. In this way tumor-associated T-helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CD8-positive T lymphocytes, CD4-positive T cells aresufficient for inhibiting manifestation of tumors via inhibition ofangiogenesis by secretion of interferon-gamma (IFNγ) (Beatty andPaterson, 2001; Mumberg et al., 1999). There is evidence for CD4 T cellsas direct anti-tumor effectors (Braumuller et al., 2013; Tran et al.,2014).

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was previously not considered possible.However, Dengjel et al. were successful in identifying a number of MHCClass II epitopes directly from tumors (WO 2007/028574, EP 1 760 088B1).

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+T cells (ligand: MHC class I molecule+peptide epitope) or byCD4-positive T-helper cells (ligand: MHC class II molecule+peptideepitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC-molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC-class-1-binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC-molecule. In this way each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T cells bearingspecific T cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or-associated antigens, and to be used in a therapy, particularprerequisites must be fulfilled. The antigen should be expressed mainlyby tumor cells and not, or in comparably small amounts, by normalhealthy tissues. In a preferred embodiment, the peptide should beover-presented by tumor cells as compared to normal healthy tissues. Itis furthermore desirable that the respective antigen is not only presentin a type of tumor, but also in high concentrations (i.e. copy numbersof the respective peptide per cell). Tumor-specific and tumor-associatedantigens are often derived from proteins directly involved intransformation of a normal cell to a tumor cell due to their function,e.g. in cell cycle control or suppression of apoptosis. Additionally,downstream targets of the proteins directly causative for atransformation may be up-regulated and thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach (Singh-Jasuja et al., 2004). It isessential that epitopes are present in the amino acid sequence of theantigen, in order to ensure that such a peptide (“immunogenic peptide”),being derived from a tumor associated antigen, and leads to an in vitroor in vivo T-cell-response.

Basically, any peptide able to bind an MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell having a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a T cellbased therapy including but not limited to tumor vaccines. The methodsfor identifying and characterizing the TAAs are usually based on the useof T-cells that can be isolated from patients or healthy subjects, orthey are based on the generation of differential transcription profilesor differential peptide expression patterns between tumors and normaltissues. However, the identification of genes over-expressed in tumortissues or human tumor cell lines, or selectively expressed in suchtissues or cell lines, does not provide precise information as to theuse of the antigens being transcribed from these genes in an immunetherapy. This is because only an individual subpopulation of epitopes ofthese antigens are suitable for such an application since a T cell witha corresponding TCR has to be present and the immunological tolerancefor this particular epitope needs to be absent or minimal. In a verypreferred embodiment of the invention it is therefore important toselect only those over- or selectively presented peptides against whicha functional and/or a proliferating T cell can be found. Such afunctional T cell is defined as a T cell, which upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T cell”).

In case of targeting peptide-MHC by specific TCRs (e.g. soluble TCRs)and antibodies or other binding molecules (scaffolds) according to theinvention, the immunogenicity of the underlying peptides is secondary.In these cases, the presentation is the determining factor.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, the present inventionrelates to a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO: 289, SEQ ID NO: 305, andSEQ ID NO: 306 or a variant sequence thereof which is at least 77%,preferably at least 88%, homologous (preferably at least 77% or at least88% identical) to SEQ ID NO: 1 to SEQ ID NO: 289, SEQ ID NO: 305, andSEQ ID NO: 306, wherein said variant binds to MHC and/or induces T cellscross-reacting with said peptide, or a pharmaceutical acceptable saltthereof, wherein said peptide is not the underlying full-lengthpolypeptide.

While the most important criterion for a peptide to function as cancertherapy target is its over-presentation on primary tumor tissues ascompared to normal tissues, also the RNA expression profile of thecorresponding gene or exon can help to select appropriate peptides.Particularly, some peptides are hard to detect by mass spectrometry,either due to their chemical properties or to their low copy numbers oncells, and a screening approach focusing on detection of peptidepresentation may fail to identify these targets. However, these targetsmay be detected by an alternative approach starting with analysis ofgene and exon expression in tumor tissues and in normal tissues andsecondarily assessing peptide presentation in tumors. This approach wasrealized in this invention using two m RNA databases (TCGA ResearchNetwork: http://cancergenome.nih.gov/ for tumor samples and GTEXdatabase (Lonsdale, 2013) for normal tissue samples), as well as peptidepresentation data. If the mRNA of a gene or exon is over-expressed intumor tissues compared to normal tissues, it is considered as tumorassociated. Such peptides, even if identified on only a small percentageof tumor tissues, represent interesting targets. Routine massspectrometry analysis is not sensitive enough to assess target coverageon the peptide level. Rather, tumor mRNA expression can be used toassess coverage. For detection of the peptide itself, a targeted massspectrometry approach with higher sensitivity than in the routinescreening may be necessary and may lead to a better estimation ofcoverage on the level of peptide presentation.

The present invention further relates to a peptide of the presentinvention comprising a sequence that is selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 289, SEQ ID NO: 305, and SEQ IDNO: 306 or a variant thereof, which is at least 77%, preferably at least88%, homologous (preferably at least 77% or at least 88% identical) toSEQ ID NO: 1 to SEQ ID NO: 289, SEQ ID NO: 305, and SEQ ID NO: 306,wherein said peptide or variant thereof has an overall length of between8 and 100, preferably between 8 and 30, and most preferred of between 8and 14 amino acids.

The present invention further relates to a peptide of the presentinvention consisting of a sequence that is selected from the groupconsisting of SEQ ID NO: 2, 24, 32, 39, 64, 72, 106, 149, 251, 305, and306.

The following tables show the peptides according to the presentinvention, their respective SEQ ID NOs, and the prospective source(underlying) genes for these peptides. All peptides in Table 3, Table 5and Table 7 (A+B) bind to HLA-A*02. All peptides in Table 4, Table 6 andTable 8 bind to HLA-A*24. The peptides in Table 5 and Table 6 have beendisclosed before in large listings as results of high-throughputscreenings with high error rates or calculated using algorithms, buthave not been associated with cancer at all before. The peptides inTable 7 (A+B) and Table 8 are additional peptides that may be useful incombination with the other peptides of the invention.

TABLE 3 Peptides according to the present invention, HLA-A*02-binding.SEQ Official ID No. Sequence GeneID(s) Gene Symbol(s)   1 LLYPEPWSV   220382 FAM181B   2 GLIAGVVSI      4233 MET   3 KLEENGDLYL     55255WDR41   4 KLMPGTYTL      2201 FBN2   5 GIVAHIQEV    440193 CCDC88C   6ALFDSLRHV    220382 FAM181B   7 ILDHEVPSL    199990 C1orf86   8SIYQFLIAV      2237 FEN1   9 FLVDGSYSI      1303 COL12A1  10 GIAGSLKTV     3720 JARID2  11 ALSPSYLTV     57674 RNF213  12 GLLPLLHRA100529261,  CHURC1-FNTB,      2342,  FNTB,     91612 CHURC1  13ALMAMLVYV     91319 DERL3  14 ILAKDLFEI     83990 BRIP1  15 YLDLSHNQL    10333, PODNL1,     79883 TLR6  16 YTLDIPVLFGV     29028 ATAD2  17AVFPDDMPTL      4297 MLL  18 ILLDLTDNRL    135228 CD109  19 SISDNVWEV    55589 BMP2K  20 GLSQITNQL      9736 USP34  21 AIQDEIRSV      4085MAD2L1  22 FVDPNTQEKV     83481 EPPK1  23 SLFSDEFKV       102 ADAM10  24TLDEKVAEL     51438 MAGEC2  25 TMDSVLVTV     94025 MUC16  26 ALQEELTEL    22995 CEP152  27 RLMEENWNA      7784 ZP3  28 SLPNGKPVSV     23682RAB38  29 YLLDPSITL     10102 TSFM  30 AMIEEVFEA    221443 OARD1  31TITETTVEV      7143 TNR  32 VQLDSIEDLEV     23532 PRAME  33 YIKTELISV     6772 STAT1  34 FLLATEVVTV     10075 HUWE1  35 FLLPFSTVYL      9204ZMYM6  36 SLADTNSLAVV      6490 PMEL  37 ILAPFSVDL     85413 SLC22A16 38 FLGPRIIGL    202309 GAPT  39 HLLEGSVGV     85320 ABCC11  40VLIDPQWVLTA      3003 GZMK  41 ALFENTPKA      5260 PHKG1  42 LLDSVSRL     3918 LAMC2  43 KAIEVLLTL     57650 KIAA1524  44 SLFETAWEA      9735KNTC1  45 SLTEVSLPL       580 BARD1  46 SQFPLPLAV     80055 PGAP1  47ALLERGELFV     79050 NOC4L  48 QVIEDSTGV     64778 FNDC3B  49 ALNIATHVL    24140 FTSJ1  50 ILFHGVFYA     55744 COA1  51 LLFSRLCGA     25945PVRL3  52 RLAVLFSGA       968 CD68  53 KMVGLVVAI     80324 PUS1  54VLNPLITAV     10827 FAM114A2  55 SLATKIVEA    152110 NEK10  56FLHDEKEGIYI     10225 CD96  57 TVFTDHMLTV       586 BCAT1  58 YLLPLLPAL   338645 LUZP2  59 KLLDPQEFTL      3371 TNC  60 ALFAPLVHL     26251KCNG2  61 AIVKEIVNI      4436 MSH2  62 ALNPELVQA      4233 MET  63SQIPAQPSV     23215 PRRC2C  64 SLFPDSLIV    261729 STEAP2  65 SVVPDVRSV     6605 SMARCE1  66 KLIFSVEAV     65985 AACS  67 TLLQRLTEV     11064CNTRL  68 SLSNRLYYL      9271 PIWIL1  69 FLAVGLVDV     28559 TRBV28  70LLLGDSALYL     28609, TRBV5-6,     28610 TRBV5-5  71 VLHSKFWVV    122618PLD4  72 FLTAINYLL    440712 C1orf186  73 YTLREVDTV      4521 NUDT1  74TLFGYSVVL      3676 ITGA4  75 AVIKFLELL      4436 MSH2  76 AVGPVHNSV    57448 BIRC6  77 TLIDEQDIPLV    116225 ZMYND19  78 TVVTRLDEI     9459 ARHGEF6  79 VTFKEYVTV      8535 CBX4  80 KLYEADFVL     55501CHST12  81 NALDKVLSV     79053 ALG8  82 FIFDEAEKL     64222 TOR3A  83GQASYFYVA      4486 MST1R  84 ALCPRIHEV      1762 DMWD  85 VLNDILVRA     5016 OVGP1  86 SVDSHFQEV      4968 OGG1  87 TIYKDFVYI     79786KLHL36  88 AQADHLPQL     64689 GORASP1  89 QLAPVFQRV     84342 COG8  90FLQDLEQRL    128272 ARHGEF19  91 KLFDESILI      8295 TRRAP  92 GLLFSLRSV    79064 TMEM223  93 QVLELDVADI      9675 TTI1  94 LLLPAVPVGA      1953MEGF6  95 GLLGSLFFL     91319 DERL3  96 LLVSHLYLV     84885 ZDHHC12  97STLPKSLSL      4605 MYBL2  98 RLFPDFFTRVAL      5657 PRTN3  99YLLQSVNQLLL     23347 SMCHD1 100 ALLGMIIVGV      6571 SLC18A2 101ALADFMLSL      2854 GPR32 102 VLLDIQEVFQI    201305 SPNS3 103 YLVSEIFKA     6005 RHAG 104 ALISWQPPRA      7143 TNR 105 ALLGTKILL    147945NLRP4 106 FINDSIVYL     89766 UMODL1 107 LLVPTSGIYFV      4049 LTA 108ILLKNLVTI    129868, TRIM43,    653192 TRIM43B 109 SLDPSVTHL       250,ALPPL2,       251 ALPP 110 FLLGVSKEV     23251 KIAA1024 111 AIVDLIHDI    23225 NUP210 112 SLGKFTFDV      6676 SPAG4 113 FLERGLESA     10535RNASEH2A 114 QLIQTLHAV      1004 CDH6 115 SLDPDTLPAV    352954, GATSL2,   389523, GATS,    729438 GATSL1 116 TIDESGSIL     29127 RACGAP1 117KMPDVELFV     79070 KDELC1 118 QLWQFLVTL      2119 ETV5 119 FIIQGLRSVGA     3117, HLA-DQA2,      3118 HLA-DQA1 120 VTPVTVSAV      3054 HCFC1121 FTIFRTISV     83743 GRWD1 122 GVVDPVHGV     83481 EPPK1 123VLDPALPALV      4486 MST1R 124 KVMATIEKV      9274 BCL7C 125 SLADYEHFV      357 SHROOM2 126 QMFQYFITV     51290 ERGIC2 127 KLDGNELDL     55379LRRC59 128 TQSPATLSV     28299, IGKV1-5,     28875, IGKV3-15,     28902,IGKV3D-15,     28913, IGKC,      3514, IGKV1D-13,     50802 IGK@ 129RLQDILWFL    150771 ITPRIPL1 130 SLLGGTFVGI     55266 TMEM19 131VTSNSGILGV     22828 SCAF8 132 ILGEVLAQL    124044 SPATA2L 133 ALLPRLHQL    85414 SLC45A3 134 GLAVPTPSV    647024 C6orf132 135 HLSTIIHEA     1147 CHUK 136 FLFGGVLMTL     91319 DERL3 137 EIASITEQL     55183RIF1 138 ALLAKILQI      5591 PRKDC 139 FLLPTGAEA      1511 CTSG 140VLLEELEAL     10142 AKAP9 141 FLDKVLVAA     54497 HEATR5B 142 ILVEGISTV     1462 VCAN 143 ALLPELREV      1140 CHRNB1 144 ALLAFFPGL     80267EDEM3 145 YLWATIQRI      2650 GCNT1 146 ALHFSEDEI      6097 RORC 147YLMDDTVEI    114327 EFHC1 148 MLAGIAITV     63826 SRR 149 ILNTHITEL   131578 LRRC15 150 VLYDRPLKI     64783 RBM15 151 SVLDSTAKV     54885TBC1D8B 152 MMVGDLLEV      5927 KDM5A 153 FISERVEVV    128869 PIGU 154RLLGTEFQV     51151 SLC45A2 155 LLNPVVEFV      5591 PRKDC 156 ILGDLSHLL    11015 KDELR3 157 TLTSLLAQA     83481 EPPK1

TABLE 4 Peptides according to the present invention, HLA-A*24-binding.SEQ Official ID No. Sequence GeneID(s) Gene Symbol(s) 158 HYSQELSLLYL     5591 PRKDC 159 LYNKGFIYL    157769 FAM91A1 160 VYTLDIPVL     29028ATAD2 161 IYLVSIPEL     23545 ATP6V0A2 162 VFTRVSSFL      1511 CTSG 163DYLKGLASF      1130 LYST 164 KFSSFSLFF      3003 GZMK 165 DYTTWTALL    10615 SPAG5 166 YYVESGKLF     23279 NUP160 167 NYINRILKL     51691NAA38 168 KYQDILETI     79730 NSUN7 169 AYTLIAPNI     94240 EPSTI1 170VYEDQVGKF     23065 EMC1 171 LFIPSSKLLFL 101060416, LOC101060416,101060589, SMG1,     23049, BOLA2,    440345, 61E3.4,    440354,LOC101060589,    552900, SMG1P1,    641298 LOC440354 172 TYTTVPRVAF    10882 C1QL1 173 IYSWILDHF      3344 FOXN2 174 VYVGGGQIIHL    151354FAM84A 175 YYEVHKELF      9055 PRC1 176 EYNQWFTKL     55604 LRRC16A 177VYPWLGALL     54905 CYP2W1 178 IFIEVFSHF    284293 HMSD 179 MYDSYWRQF   143686 SESN3 180 IYDDSFIRPVTF     56886 UGGT1 181 LYLDIINLF     51643TMBIM4 182 IYQLDTASI     55003 PAK1IP1 183 VFTSTARAF     10225 CD96 184VFQNFPLLF     56890 MDM1 185 IYKVGAPTI     10978 CLP1 186 IFPQFLYQF    23250 ATP11A 187 TYLRDQHFL       898 CCNE1 188 RYFKGLVF    166614DCLK2 189 WYVNGVNYF     79054 TRPM8 190 GFFIFNERF     10206 TRIM13 191VFKASKITF      5803 PTPRZ1 192 SYALLTYMI      7298 TYMS 193 RFHPTPLLL    51605 TRMT6 194 EFGSLHLEFL     57134 MAN1C1 195 TYSVSFPMF    257202,GPX6,      2882 GPX7 196 LYIDRPLPYL    253725, FAM21C,    387680,FAM21B,     55747 FAM21A 197 EYSLFPGQVVI     23649 POLA2 198 LYLDKATLI    55916 NXT2 199 RYAEEVGIF     54187 NANS 200 YYGPSLFLL     10075HUWE1 201 IYATEAHVF     55706 TMEM48 202 VYWDSAGAAHF     55501 CHST12203 FYSRLLQKF     55055 ZWILCH 204 TYELRYFQI    200916 RPL22L1 205VHIPEVYLI     57705 WDFY4 206 EYQENFLSF    441027 TMEM150C 207 AYVVFVSTL    25938 HEATR5A 208 TYTQDFNKF       796 CALCA 209 TYKDEGNDYF      7268TTC4 210 IYTMIYRNL      4193 MDM2 211 YYLEVGKTLI     80267 EDEM3 212YYTFHFLYF     26273 FBXO3 213 IFDEAEKL     64222 TOR3A 214 LYLKLWNLI    11274, USP18,    373856 USP41 215 YFDKVVTL    374654 KIF7 216QYSSVFKSL     25938 HEATR5A 217 FFPPTRQMGLLF     85415 RHPN2 218YYKSTSSAF     79690 GAL3ST4 219 EYPLVINTL 100129460, DPY19L1P1,    23333 DPY19L1 220 GYIDNVTLI      3918 LAMC2 221 RYSTGLAGNLL    54921 CHTF8 222 TFSVSSHLF     90874 ZNF697 223 KYIPYKYVI     57674RNF213 224 QYLENLEKL 101060589, 61E3.4,     23049, SMG1P1,    440345,SMG1,    641298 LOC101060589 225 YYVYIMNHL     10655 DMRT2 226VYRDETGELF     84455 EFCAB7 227 IFLDYEAGTLSF    202658, TRIM39,    56658 TRIM39-RPP21 228 KYTSWYVAL      2247 FGF2

TABLE 5 Additional peptides according to thepresent invention with no prior known cancerassociation, HLA-A*02-binding. SEQ Official ID No. Sequence GeneID(s)Gene Symbol(s) 229 AILAHLNTV   8914 TIMELESS 230 KLQNIMMLL   3070 HELLS231 MLDKYSHYL 139818 DOCK11 232 KIFPAALQLV   2618 GART 233 HLFDAFVSV 55789 DEPDC1B 234 LLSPHNPAL  10884 MRPS30 235 KIIDFLSAL   2956 MSH6 236STIAILNSV  23310 NCAPD3 237 ALAPHLDDA  54919 HEATR2 238 GLYERPTAA   2672GFI1 239 KMNESTRSV   4173 MCM4 240 YMGEEKLIASV  23404 EXOSC2 241KTIQQLETV  55127 HEATR1 242 WLYGEDHQI    586 BCAT1 243 FMADDIFSV  50615IL21R 244 YLLEKNRVV   4651 MYO10 245 SLLDLPLSL   9487 PIGL 246 TVSDVLNSV 22796 COG2 247 ALYEGYATV  10072, DPP3,    582 BBS1 248 YLDRFLAGV    894CCND2 249 GLCERLVSL   5591 PRKDC 250 SLAPATPEV   9308 CD83 251 ALSVLRLAL  6691 SPINK2 252 RLMEICESL   1063 CENPF 253 ALAELIDNSL  23347 SMCHD1254 KLQGKLPEL   5198 PFAS 255 SLLHFTENL 157570 ESCO2 256 SLGEEQFSV157570 ESCO2 257 GLYTDPCGV  55007 FAM118A 258 LLSERFINV  56647 BCCIP 259ILLPRIIEA  83959 SLC4A11 260 ILLEKILSL   9373 PLAA 261 QLQDRVYAL  55374TMCO6 262 FMVDKAIYL  55068 ENOX1 263 VLLSEQGDVKL  10494, STK25,  51765,MST4,   6788 STK3 264 KLFPQETLFL  11124 FAF1 265 NTCPYVHNI  51728 POLR3K266 YAIGLVMRL 401494 PTPLAD2

TABLE 6 Additional peptides according to thepresent invention with no prior known cancerassociation, HLA-A*24-binding. SEQ Official ID No. Sequence GeneID(s)Gene Symbol(s) 267 KYMVYPQTF  10884 MRPS30 268 QYLGQIQHI   7298 TYMS 269YFIDSTNLKTHF  51042 ZNF593 270 NYYEVHKELF   9055 PRC1 271 LYHDIFSRL  9603 NFE2L3 272 QYLQDAYSF   9918 NCAPD2 273 TYIKPISKL   4644 MYO5A 274AYLHSHALI  51347 TAOK3 275 EYINQGDLHEF   4919 ROR1 276 VYGFQWRHF   7298TYMS 277 VYQGHTALL   5754 PTK7 278 RYISDQLFTNF  23268 DNMBP 279TYIESASEL  79623 GALNT14 280 RYPDNLKHLYL  29080 CCDC59 281 PYRLIFEKF  5591 PRKDC 282 KFVDSTFYL   9688 NUP93 283 TYGDAGLTYTF 121642 ALKBH2284 RYLNKAFHI  23310 NCAPD3 285 HYPPVQVLF   2956 MSH6 286 RYPDNLKHL 29080 CCDC59 287 LYITEPKTI  11219, TREX2,  55559 HAUS7 288 VYVSDIQEL 23225, NUP210P1, 255330 NUP210 289 KYPVEWAKF  51101 ZC2HC1A

TABLE 7A Peptides useful for e.g. personalizedcancer therapies, HLA-A*02-binding. SEQ Official ID No. SequenceGeneID(s) Gene Symbol(s) 290 KIVDFSYSV   701 BUB1B 291 KLDETGNSL  7153TOP2A 292 GMMTAILGV 79939 SLC35E1 293 FLVDGSWSI 57642 COL20A1 294GLMKYIGEV 79054 TRPM8

TABLE 7B Peptides useful for e.g. personalizedcancer therapies, HLA-A*02-binding. SEQ Official ID No. SequenceGeneID(s) Gene Symbol(s) 305 KLFTSVFGV 1791 DNTT 306 ALLSSLNEL  367 AR

TABLE 8 Peptides useful for e.g. personalizedcancer therapies, HLA-A*24-binding. SEQ Official ID No. SequenceGeneID(s) Gene Symbol(s) 295 YYPGVILGF 55026 TMEM255A 296 TYVDSSHTI 1462 VCAN 297 PFLQASPHF 84985 FAM83A 298 RYLEGTSCI 83481 EPPK1 299VYFVAPAKF  3918 LAMC2 300 AYVLRLETL 10687 PNMA2 301 AYKPGALTF 84883AIFM2 302 RYMPPAHRNF  3620 IDO1

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1T describe relative presentation of various peptides of thepresent disclosure.

FIGS. 2A-2I describe relative group of various peptides of the presentdisclosure.

FIGS. 3A and 3B describe simulation graphs of various peptides of thepresent disclosure.

FIGS. 4A-4D describe simulation graphs of various peptides of thepresent disclosure.

FIGS. 5A-5C describe simulation graphs of various peptides of thepresent disclosure.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

The present invention furthermore generally relates to the peptidesaccording to the present invention for use in the treatment ofproliferative diseases, such as, for example, glioblastoma (GB), breastcancer (BRCA), colorectal cancer (CRC), renal cell carcinoma (RCC),chronic lymphocytic leukemia (CLL), hepatocellular carcinoma (HCC),non-small cell and small cell lung cancer (NSCLC, SCLC), Non-Hodgkinlymphoma (NHL), acute myeloid leukemia (AML), ovarian cancer (OC),pancreatic cancer (PC), prostate cancer (PCA), esophageal cancerincluding cancer of the gastric-esophageal junction (OSCAR), gallbladdercancer and cholangiocarcinoma (GBC, CCC), melanoma (MEL), gastric cancer(GC), urinary bladder cancer (UBC), head- and neck squamous cellcarcinoma (HNSCC), and uterine cancer (UEC).

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 289, SEQ ID NO: 305, and SEQ IDNO: 306. More preferred are the peptides—alone or incombination—selected from the group consisting of SEQ ID NO: 1 to SEQ IDNO: 149 (see Table 3), SEQ ID NO: 158 to SEQ ID NO: 213 (see Table 4),and SEQ ID NO: 305, and SEQ ID NO: 306, in particular consisting of asequence that is selected from the group consisting of SEQ ID NO: 2, 24,32, 39, 64, 72, 106, 149, 251, 305, and 306, and their uses in theimmunotherapy of glioblastoma, breast cancer, colorectal cancer, renalcell carcinoma, chronic lymphocytic leukemia, hepatocellular carcinoma,non-small cell and small cell lung cancer, Non-Hodgkin lymphoma, acutemyeloid leukemia, ovarian cancer, pancreatic cancer, prostate cancer,esophageal cancer including cancer of the gastric-esophageal junction,gallbladder cancer and cholangiocarcinoma, melanoma, gastric cancer,urinary bladder cancer, head- and neck squamous cell carcinoma (HNSCC),or uterine cancer.

As shown in Example 1, many of the peptides according to the presentinvention are found on various tumor types and can, thus, be used in theimmunotherapy of other indications. Over-expression of the underlyingpolypeptides in a variety of cancers, as shown in Example 2, hintstowards the usefulness of these peptides in various other oncologicalindications.

Thus, another aspect of the present invention relates to the use of thepeptides according to the present invention for the—preferablycombined—treatment of a proliferative disease selected from the group ofglioblastoma, breast cancer, colorectal cancer, renal cell carcinoma,chronic lymphocytic leukemia, hepatocellular carcinoma, non-small celland small cell lung cancer, Non-Hodgkin lymphoma, acute myeloidleukemia, ovarian cancer, pancreatic cancer, prostate cancer, esophagealcancer including cancer of the gastric-esophageal junction, gallbladdercancer and cholangiocarcinoma, melanoma, gastric cancer, urinary bladdercancer, head- and neck squamous cell carcinoma, or uterine cancer.

The present invention furthermore relates to peptides according to thepresent invention that have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or—in an elongatedform, such as a length-variant—MHC class-II.

The present invention further relates to the peptides according to thepresent invention wherein said peptides (each) consist or consistessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 289, SEQ ID NO: 305, and SEQ ID NO: 306.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to the presentinvention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use in thetreatment of diseases and in medicine, in particular in the treatment ofcancer.

The present invention further relates to antibodies that are specificagainst the peptides according to the present invention or complexes ofsaid peptides according to the present invention with MHC, and methodsof making these.

The present invention further relates to T-cell receptors (TCRs), inparticular soluble TCR (sTCRs) and cloned TCRs engineered intoautologous or allogeneic T cells, and methods of making these, as wellas NK cells or other cells bearing said TCR or cross-reacting with saidTCRs.

The antibodies and TCRs are additional embodiments of theimmunotherapeutic use of the peptides according to the invention athand.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell, and preferably is a dendritic cell.

The present invention further relates to a method for producing apeptide according to the present invention, said method comprisingculturing the host cell according to the present invention, andisolating the peptide from said host cell or its culture medium.

The present invention further relates to said method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing or expressing said peptidecontaining SEQ ID No. 1 to SEQ ID No.: 289, preferably containing SEQ IDNO: 1 to SEQ ID NO: 149, SEQ ID NO: 158 to SEQ ID NO: 213, or a variantamino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellselectively recognizes a cell which expresses a polypeptide comprisingan amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as produced according to the present invention.

The present invention further relates to the use of any peptide asdescribed, the nucleic acid according to the present invention, theexpression vector according to the present invention, the cell accordingto the present invention, the activated T lymphocyte, the T cellreceptor or the antibody or other peptide- and/or peptide-MHC-bindingmolecules according to the present invention as a medicament or in themanufacture of a medicament. Preferably, said medicament is activeagainst cancer.

Preferably, said medicament is a cellular therapy, a vaccine or aprotein based on a soluble TCR or antibody.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are glioblastoma, breast cancer,colorectal cancer, renal cell carcinoma, chronic lymphocytic leukemia,hepatocellular carcinoma, non-small cell and small cell lung cancer,Non-Hodgkin lymphoma, acute myeloid leukemia, ovarian cancer, pancreaticcancer, prostate cancer, esophageal cancer including cancer of thegastric-esophageal junction, gallbladder cancer and cholangiocarcinoma,melanoma, gastric cancer, urinary bladder cancer, head- and necksquamous cell carcinoma, or uterine cancer cells.

The present invention further relates to biomarkers based on thepeptides according to the present invention, herein called “targets”,that can be used in the diagnosis of cancer, preferably glioblastoma,breast cancer, colorectal cancer, renal cell carcinoma, chroniclymphocytic leukemia, hepatocellular carcinoma, non-small cell and smallcell lung cancer, Non-Hodgkin lymphoma, acute myeloid leukemia, ovariancancer, pancreatic cancer, prostate cancer, esophageal cancer includingcancer of the gastric-esophageal junction, gallbladder cancer andcholangiocarcinoma, melanoma, gastric cancer, urinary bladder cancer,head- and neck squamous cell carcinoma, or uterine cancer. The markercan be over-presentation of the peptide(s) themselves, orover-expression of the corresponding gene(s). The markers may also beused to predict the probability of success of a treatment, preferably animmunotherapy, and most preferred an immunotherapy targeting the sametarget that is identified by the biomarker. For example, an antibody orsoluble TCR can be used to stain sections of the tumor to detect thepresence of a peptide of interest in complex with MHC.

Optionally the antibody carries a further effector function such as animmune stimulating domain or toxin.

The present invention also relates to the use of these targets in thecontext of cancer treatment.

A single nucleotide polymorphism of ABCC11 was shown to be associatedwith a shorter relapse-free survival in patients with non-small celllung cancer who were treated with S-1 adjuvant chemotherapy (Tsuchiya etal., 2016). ABCC11 was described as a promoter of a multi-drugresistance phenotype in breast cancer. Furthermore, high expression ofABCC11 in breast tumors was shown to be associated with aggressivesubtypes and low disease-free survival (Honorat et al., 2013; Yamada etal., 2013). ABCC11 transcript levels in colorectal cancer patients wereshown to be significantly lower in non-responders to palliativechemotherapy in comparison with responders which associated withsignificantly shorter disease-free intervals (Hlavata et al., 2012).ABCC11 was described as a potential biomarker for pemetrexed (MTA)treatment in lung adenocarcinomas (Uemura et al., 2010). ABCC11up-regulation in acute myeloid leukemia was shown to be associated witha low probability of overall survival assessed over 4 years and mayserve as a predictive marker (Guo et al., 2009). ABCC11 was shown to beup-regulated in hepatocellular carcinoma (Borel et al., 2012).

AR encodes for the androgen receptor gene which is more than 90 kb longand codes for a protein that has 3 major functional domains: theN-terminal domain, DNA-binding domain, and androgen-binding domain. Theprotein functions as a steroid-hormone activated transcription factor.Upon binding the hormone ligand, the receptor dissociates from accessoryproteins, translocates into the nucleus, dimerizes, and then stimulatestranscription of androgen responsive genes. This gene contains 2polymorphic trinucleotide repeat segments that encode polyglutamine andpolyglycine tracts in the N-terminal transactivation domain of itsprotein. Expansion of the polyglutamine tract from the normal 9-34repeats to the pathogenic 38-62 repeats causes spinal bulbar muscularatrophy (Kennedy disease). Mutations in this gene are also associatedwith complete androgen insensitivity (CAIS). Two alternatively splicedvariants encoding distinct isoforms have been described. US20150306197A1 discloses SQ ID NO. 305 as an AR LBD (ligand-binding domain) peptideepitope that was identified by scanning the protein sequence of the ARLBD for 9-mer or 10-mer peptides that fit the HLA-A2 consensus bindingsequence and by their predicted binding affinity to HLA-A2. The peptideis exclusively proposed for a prostate cancer vaccine.

C1orf186 encodes chromosome 1 open reading frame 186 and is located onchromosome 1₈32.1 (RefSeq, 2002). Krüppel-like factor 9 inhibitsC1orf186 expression in endometrial carcinoma cells (Simmen et al.,2008). C1orf186 is associated with ER-positive breast cancer (Triulzi etal., 2015).

DNTT encodes for DNA nucleotidylexotransferase. In vivo, the encodedprotein is expressed in a restricted population of normal and malignantpre-B and pre-T lymphocytes during early differentiation, where itgenerates antigen receptor diversity by synthesizing non-germ lineelements (N-regions) at the junctions of rearranged Ig heavy chain and Tcell receptor gene segments. Alternatively spliced transcript variantsencoding different isoforms of this gene have been described.US20110142842 A1 predicts the peptide of SEQ ID NO. 305 as binding toHLA-A*0201 as a sequences of hematopoietic cell-specific proteins. Thepeptide is not tested further, and the publication speculates about thetreatment of many different types of cancer including leukemia,lymphomas such as non-Hodgkin lymphoma and multiple myeloma.

LRRC15 encodes leucine rich repeat containing 15 and is located onchromosome 3q29 (RefSeq, 2002). EWSR1-WT1 is an oncogenic transcriptionfactor that was shown to affect the expression of LRRC15 (Cliteur etal., 2012; Reynolds et al., 2003). LRRC15 is a tumor antigen which isover-expressed in a variety of entities (O'Prey et al., 2008). LRRC15 isassociated with breast cancer invasion (Schuetz et al., 2006). LRRC15 isassociated with aggressive behavior of androgen-independent metastaticprostate tumors (Stanbrough et al., 2006). Autoantibodies against LRRC15are inversely correlated with breast cancer (Evans et al., 2014).

MAGEC2 encodes MAGE family member C2, a gene clustered on chromosomeXq26-q27 like the other MAGEC genes (RefSeq, 2002). Over-expression ofMAGEC2 increases the level of cyclin E and promotes G1-S transition andcell proliferation (Hao et al., 2015). MAGEC2 promotes proliferation andresistance to apoptosis in Multiple Myeloma suggesting thatMAGEC2-specific immunotherapies have the potential to eradicate the mostmalignant cells (Lajmi et al., 2015). MAGEC2, an epithelial-mesenchymaltransition inducer, is associated with breast cancer metastasis.Multivariate analyses showed that MAGEC2 expression was an independentrisk factor for patient overall survival and metastasis-free survival(Yang et al., 2014).

MET encodes the hepatocyte growth factor receptor and encodestyrosine-kinase activity (RefSeq, 2002). MET was shown to beup-regulated in dedifferentiated liposarcoma and is associated withmelanocytic tumors, hepatocellular carcinoma, non-small cell lungcancer, hereditary papillary kidney cancers and gastric adenocarcinomas(Petrini, 2015; Finocchiaro et al., 2015; Steinway et al., 2015; Bill etal., 2015; Yeh et al., 2015).

PRAME encodes an antigen that is preferentially expressed in humanmelanomas and acts as a repressor of retinoic acid receptor, likelyconferring a growth advantage to cancer cell via this function (RefSeq,2002). PRAME was shown to be up-regulated in multiple myeloma, clearcell renal cell carcinoma, breast cancer, acute myeloid leukemia,melanoma, chronic myeloid leukemia, head and neck squamous cellcarcinoma and osteosarcoma cell lines (Dannenmann et al., 2013; Yao etal., 2014; Zou et al., 2012; Szczepanski and Whiteside, 2013; Zhang etal., 2013; Beard et al., 2013; Abdelmalak et al., 2014; Qin et al.,2014). PRAME is associated with myxoid and round-cell liposarcoma(Hemminger et al., 2014). PRAME is associated with shorterprogression-free survival and chemotherapeutic response in diffuse largeB-cell lymphoma treated with R-CHOP, markers of poor prognosis in headand neck squamous cell carcinoma, poor response to chemotherapy inurothelial carcinoma and poor prognosis and lung metastasis inosteosarcoma (Tan et al., 2012; Dyrskjot et al., 2012; Szczepanski etal., 2013; Mitsuhashi et al., 2014). PRAME is associated with lowerrelapse, lower mortality and overall survival in acute lymphoblasticleukemia (Abdelmalak et al., 2014). PRAME may be a prognostic marker fordiffuse large B-cell lymphoma treated with R-CHOP therapy (Mitsuhashi etal., 2014).

SPINK2 encodes a member of the family of serine protease inhibitors ofthe Kazal type which acts as a trypsin and acrosin inhibitor in thegenital tract and is localized in the spermatozoa (RefSeq, 2002). SPINK2was shown to be significantly up-regulated in most leukemia cell linesexcept B-lymphoblast TK-6 cells, and was suggested to play an importantrole in tumor progression and response to treatment (Chen et al., 2009).

STEAP2 encodes STEAP2 metalloreductase which encodes a multi-passmembrane protein that localizes to the Golgi complex, the plasmamembrane, and the vesicular tubular structures in the cytosol. Increasedtranscriptional expression of the human gene is associated with prostatecancer progression (RefSeq, 2002). STEAP2 is induced upon TNF-alpha andrepressed upon NF-kappaB treatment. Silencing of NF-kappaB leads to anover-expression of the anti-apoptotic protein STEAP2 which subsequentlyrepresses p53 (Gonen-Korkmaz et al., 2014). STEAP2 is over-expressed inmany cancer entities like prostate, bladder, colon, pancreas, ovary,testis, breast, cervix, and Ewing sarcoma (Wang et al., 2010; Gomes etal., 2012; Grunewald et al., 2012). STEAP2 may drive prostate cancercell migration and invasion. Over-expression of STEAP2 is associatedwith locally advanced disease state (Whiteland et al., 2014). STEAP2 hasa greater proportion of unspliced RNA in castration-resistant prostatecancer (Sowalsky et al., 2015). STEAP2 can be used as biomarker forprostate cancer (Edwards et al., 2005). STEAP2 is associated withsimvastatin and lovastatin resistance (Savas et al., 2011).

UMODL1 encodes uromodulin like 1 and is located on chromosome 21q22.3(RefSeq, 2002). UMODL1 may drive lung adenocarcinoma metastasis (Tan etal., 2016). A long non-coding RNA chimera, UMODL1-AS1, can be used asprognostic factor for breast cancer recurrence (Liu et al., 2016).

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defect ribosomal products (DRIPS) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

As used herein and except as noted otherwise all terms are defined asgiven below.

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, or 12 or longer, and incase of MHC class II peptides (elongated variants of the peptides of theinvention) they can be as long as 13, 14, 15, 16, 17, 18, 19 or 20 ormore amino acids in length.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutical acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present invention differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not salts invivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thelength of the oligopeptide is not critical to the invention, as long asthe correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 15 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it.

A class I T cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by a Tcell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-14 amino acids in length, and most typically 9amino acids in length.

In humans there are three different genetic loci that encode MHC class Imolecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

TABLE 9 Expression frequencies F of HLA-A*02 and HLA-A*24 and the mostfrequent HLA-DR serotypes. Frequencies are deduced from haplotypefrequencies Gf within the American population adapted from Mori et al.(Mori et al., 1997) employing the Hardy-Weinberg formula F = 1 − (1 −Gf)². Combinations of A*02 or A*24 with certain HLA-DR alleles might beenriched or less frequent than expected from their single frequenciesdue to linkage disequilibrium. For details refer to Chanock et al.(Chanock et al., 2004). Calculated phenotype Allele Population fromallele frequency A*02 Caucasian (North America)  49.1% A*02 AfricanAmerican (North America)  34.1% A*02 Asian American (North America) 43.2% A*02 Latin American (North American)  48.3% DR1 Caucasian (NorthAmerica)  19.4% DR2 Caucasian (North America)  28.2% DR3 Caucasian(North America)  20.6% DR4 Caucasian (North America)  30.7% DR5Caucasian (North America)  23.3% DR6 Caucasian (North America)  26.7%DR7 Caucasian (North America)  24.8% DR8 Caucasian (North America)  5.7%DR9 Caucasian (North America)  2.1% DR1 African (North) American 13.20%DR2 African (North) American 29.80% DR3 African (North) American 24.80%DR4 African (North) American 11.10% DR5 African (North) American 31.10%DR6 African (North) American 33.70% DR7 African (North) American 19.20%DR8 African (North) American 12.10% DR9 African (North) American  5.80%DR1 Asian (North) American  6.80% DR2 Asian (North) American 33.80% DR3Asian (North) American  9.20% DR4 Asian (North) American 28.60% DR5Asian (North) American 30.00% DR6 Asian (North) American 25.10% DR7Asian (North) American 13.40% DR8 Asian (North) American 12.70% DR9Asian (North) American 18.60% DR1 Latin (North) American 15.30% DR2Latin (North) American 21.20% DR3 Latin (North) American 15.20% DR4Latin (North) American 36.80% DR5 Latin (North) American 20.00% DR6Latin (North) American 31.10% DR7 Latin (North) American 20.20% DR8Latin (North) American 18.60% DR9 Latin (North) American  2.10% A*24Philippines   65% A*24 Russia Nenets   61% A*24:02 Japan   59% A*24Malaysia   58% A*24:02 Philippines   54% A*24 India   47% A*24 SouthKorea   40% A*24 Sri Lanka   37% A*24 China   32% A*24:02 India   29%A*24 Australia West   22% A*24 USA   22% A*24 Russia Samara   20% A*24South America   20% A*24 Europe   18%

The peptides of the invention, preferably when included into a vaccineof the invention as described herein bind to A*02 or A*24. A vaccine mayalso include pan-binding MHC class II peptides. Therefore, the vaccineof the invention can be used to treat cancer in patients that are A*02or A*24 positive, whereas no selection for MHC class II allotypes isnecessary due to the pan-binding nature of these peptides.

If A*02 peptides of the invention are combined with peptides binding toanother allele, for example A*24, a higher percentage of any patientpopulation can be treated compared with addressing either MHC class Iallele alone. While in most populations less than 50% of patients couldbe addressed by either allele alone, a vaccine comprising HLA-A*24 andHLA-A*02 epitopes can treat at least 60% of patients in any relevantpopulation. Specifically, the following percentages of patients will bepositive for at least one of these alleles in various regions: USA 61%,Western Europe 62%, China 75%, South Korea 77%, Japan 86%.

In a preferred embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly encompassed.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform. The term “active fragment” means a fragment, usually of a peptide,polypeptide or nucleic acid sequence, that generates an immune response(i.e., has immunogenic activity) when administered, alone or optionallywith a suitable adjuvant or in a vector, to an animal, such as a mammal,for example, a rabbit or a mouse, and also including a human, suchimmune response taking the form of stimulating a T-cell response withinthe recipient animal, such as a human. Alternatively, the “activefragment” may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment”, when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The percent identity isthen determined according to the following formula:percent identity=100[1−(C/R)]

wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein

(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and

(ii) each gap in the Reference Sequence and

(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and

(iiii) the alignment has to start at position 1 of the alignedsequences;

and R is the number of bases or amino acids in the Reference Sequenceover the length of the alignment with the Compared Sequence with any gapcreated in the Reference Sequence also being counted as a base or aminoacid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity.

As mentioned above, the present invention thus provides a peptidecomprising a sequence that is selected from the group of consisting ofSEQ ID NO: 1 to SEQ ID NO: 289 or a variant thereof which is 88%homologous to SEQ ID NO: 1 to SEQ ID NO: 289, or a variant thereof thatwill induce T cells cross-reacting with said peptide. The peptides ofthe invention have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or elongated versions of saidpeptides to class II.

In the present invention, the term “homologous” refers to the degree ofidentity (see percent identity above) between sequences of two aminoacid sequences, i.e. peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Appay et al., 2006; Colombetti et al., 2006;Fong et al., 2001; Zaremba et al., 1997).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 289. Forexample, a peptide may be modified so that it at least maintains, if notimproves, the ability to interact with and bind to the binding groove ofa suitable MHC molecule, such as HLA-A*02 or -DR, and in that way it atleast maintains, if not improves, the ability to bind to the TCR ofactivated T cells.

These T cells can subsequently cross-react with cells and kill cellsthat express a polypeptide that contains the natural amino acid sequenceof the cognate peptide as defined in the aspects of the invention. Ascan be derived from the scientific literature and databases (Rammenseeet al., 1999; Godkin et al., 1997), certain positions of HLA bindingpeptides are typically anchor residues forming a core sequence fittingto the binding motif of the HLA receptor, which is defined by polar,electrophysical, hydrophobic and spatial properties of the polypeptidechains constituting the binding groove. Thus, one skilled in the artwould be able to modify the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO: 289, 305, and 306, by maintaining the known anchorresidues, and would be able to determine whether such variants maintainthe ability to bind MHC class I or II molecules. The variants of thepresent invention retain the ability to bind to the TCR of activated Tcells, which can subsequently cross-react with and kill cells thatexpress a polypeptide containing the natural amino acid sequence of thecognate peptide as defined in the aspects of the invention.

The original (unmodified) peptides as disclosed herein can be modifiedby the substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.Preferably those substitutions are located at the end of the amino acidchain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,non-standard amino acids (i.e., other than the common naturallyoccurring proteinogenic amino acids) may also be used for substitutionpurposes to produce immunogens and immunogenic polypeptides according tothe present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would be simultaneously substituted.

A peptide consisting essentially of the amino acid sequence as indicatedherein can have one or two non-anchor amino acids (see below regardingthe anchor motif) exchanged without that the ability to bind to amolecule of the human major histocompatibility complex (MHC) class-I or-II is substantially changed or is negatively affected, when compared tothe non-modified peptide. In another embodiment, in a peptide consistingessentially of the amino acid sequence as indicated herein, one or twoamino acids can be exchanged with their conservative exchange partners(see herein below) without that the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or -II issubstantially changed, or is negatively affected, when compared to thenon-modified peptide.

The amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith other amino acids whose incorporation do not substantially affectT-cell reactivity and does not eliminate binding to the relevant MHC.Thus, apart from the proviso given, the peptide of the invention may beany peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 10 Variants and motif of the HLA-A*02-binding peptides accordingto SEQ ID NO: 1, 2, and 3. Position 1 2 3 4 5 6 7 8 9 10 SEQ ID NO. 1 LL Y P E P W S V Variant I L A M M I M L M A A A I A L A A V V I V L V AT T I T L T A Q Q I Q L Q A SEQ ID NO. 2 G L I A G V V S I Variant V L AM V M M L M A A V A A L A A V V V V L V A T V T T L T A Q V Q Q L Q ASEQ ID NO. 3 K L E E N G D L Y L Variant V I A M V M I M M A A V A I A AA V V V I V V A T V T I T T A Q V Q I Q

TABLE 11 Variants and motif of the HLA-A*24-binding peptides accordingto SEQ ID NO: 158, 159, and 160. Position 1 2 3 4 5 6 7 8 9 10 11SEQ ID NO. 158 H Y S Q E L S L L Y L Variant I F F I F F FSEQ ID NO. 159 L Y N K G F I Y L Variant I F F I F F F SEQ ID NO. 160 VY T L D I P V L Variant I F F I F F F

Longer (elongated) peptides may also be suitable. It is possible thatMHC class I epitopes, although usually between 8 and 11 amino acidslong, are generated by peptide processing from longer peptides orproteins that include the actual epitope. It is preferred that theresidues that flank the actual epitope are residues that do notsubstantially affect proteolytic cleavage necessary to expose the actualepitope during processing.

The peptides of the invention can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 4:0 and 0:4. Combinations of the elongationsaccording to the invention can be found in Table 12.

TABLE 12 Combinations of the elongations of peptides of the inventionC-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

In an alternative embodiment, the peptide is elongated on either or bothsides by more than 4 amino acids, preferably to a total length of up to30 amino acids. This may lead to MHC class II binding peptides. Bindingto MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHCclass I epitopes, wherein the peptide or variant has an overall lengthof between 8 and 100, preferably between 8 and 30, and most preferredbetween 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids, in caseof the elongated class II binding peptides the length can also be 15,16, 17, 18, 19, 20, 21 or 22 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to a MHC complex may be tested by methods known in the art.

Preferably, when the T cells specific for a peptide according to thepresent invention are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 μM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 pM, andmost preferably no more than about 10 pM. It is also preferred that thesubstituted peptide be recognized by T cells from more than oneindividual, at least two, and more preferably three individuals.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 289, 305, and 306.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO: 289, 305, and 306 or a variant thereof containsadditional N- and/or C-terminally located stretches of amino acids thatare not necessarily forming part of the peptide that functions as anepitope for MHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is partof a fusion protein which comprises, for example, the 80 N-terminalamino acids of the HLA-DR antigen-associated invariant chain (p33, inthe following “Ii”) as derived from the NCBI, GenBank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular into a sequence of an antibody, so as to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for dendritic cells as described herein.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) (Meziere et al., 1997),incorporated herein by reference. This approach involves makingpseudopeptides containing changes involving the backbone, and not theorientation of side chains. Meziere et al. (Meziere et al., 1997) showthat for MHC binding and T helper cell responses, these pseudopeptidesare useful. Retro-inverse peptides, which contain NH—CO bonds instead ofCO—NH peptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well-knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2004 (Lundblad, 2004), which isincorporated herein by reference. Chemical modification of amino acidsincludes but is not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995)for more extensive methodology relating to chemical modification ofproteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich(http://www.sigma-aldrich.com) provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals. Woodward's Reagent K may be used to modify specificglutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimidecan be used to form intra-molecular crosslinks between a lysine residueand a glutamic acid residue. For example, diethylpyrocarbonate is areagent for the modification of histidyl residues in proteins. Histidinecan also be modified using 4-hydroxy-2-nonenal. The reaction of lysineresidues and other α-amino groups is, for example, useful in binding ofpeptides to surfaces or the cross-linking of proteins/peptides. Lysineis the site of attachment of poly(ethylene)glycol and the major site ofmodification in the glycosylation of proteins. Methionine residues inproteins can be modified with e.g. iodoacetamide, bromoethylamine, andchloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modificationof tyrosyl residues. Cross-linking via the formation of dityrosine canbe accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethylene glycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention.

Another embodiment of the present invention relates to a non-naturallyoccurring peptide wherein said peptide consists or consists essentiallyof an amino acid sequence according to SEQ ID No: 1 to SEQ ID No: 289,SEQ ID NO: 305, and SEQ ID NO: 306 and has been synthetically produced(e.g. synthesized) as a pharmaceutically acceptable salt. Methods tosynthetically produce peptides are well known in the art. The salts ofthe peptides according to the present invention differ substantiallyfrom the peptides in their state(s) in vivo, as the peptides asgenerated in vivo are no salts. The non-natural salt form of the peptidemediates the solubility of the peptide, in particular in the context ofpharmaceutical compositions comprising the peptides, e.g. the peptidevaccines as disclosed herein. A sufficient and at least substantialsolubility of the peptide(s) is required in order to efficiently providethe peptides to the subject to be treated. Preferably, the salts arepharmaceutically acceptable salts of the peptides. These salts accordingto the invention include alkaline and earth alkaline salts such as saltsof the Hofmeister series comprising as anions PO₄ ³⁻, SO₄ ²⁻, CH₃COO⁻,Cl⁻, Br⁻, NO₃ ⁻, ClO₄ ⁻, I⁻, SCN⁻ and as cations NH₄ ⁺, Rb⁺, K⁺, Na⁺,Cs⁺, Li⁺, Zn²⁺, Mg²⁺, Ca²⁺, Mn²⁺, Cu²⁺ and Ba²⁺. Particularly salts areselected from (NH₄)₃PO₄, (NH₄)₂HPO₄, (NH₄)H₂PO₄, (NH₄)₂SO₄, NH₄CH₃COO,NH₄Cl, NH₄Br, NH₄NO₃, NH₄ClO₄, NH₄I, NH₄SCN, Rb₃PO₄, Rb₂HPO₄, RbH₂PO₄,Rb₂SO₄, Rb₄CH₃COO, Rb₄Cl, Rb₄Br, Rb₄NO₃, Rb₄ClO₄, Rb₄I, Rb₄SCN, K₃PO₄,K₂HPO₄, KH₂PO₄, K₂SO₄, KCH₃COO, KCl, KBr, KNO₃, KClO₄, KI, KSCN, Na₃PO₄,Na₂HPO₄, NaH₂PO₄, Na₂SO₄, NaCH₃COO, NaCl, NaBr, NaNO₃, NaClO₄, NaI,NaSCN, ZnCl₂ Cs₃PO₄, Cs₂HPO₄, CsH₂PO₄, Cs₂SO₄, CsCH₃COO, CsCl, CsBr,CsNO₃, CsClO₄, CsI, CsSCN, Li₃PO₄, Li₂HPO₄, LiH₂PO₄, Li₂SO₄, LiCH₃COO,LiCl, LiBr, LiNO₃, LiClO₄, LiI, LiSCN, Cu₂SO₄, Mg₃(PO₄)₂, Mg₂HPO₄,Mg(H₂PO₄)₂, Mg₂SO₄, Mg(CH₃COO)₂, MgCl₂, MgBr₂, Mg(NO₃)₂, Mg(ClO₄)₂,MgI₂, Mg(SCN)₂, MnCl₂, Ca₃(PO₄), Ca₂HPO₄, Ca(H₂PO₄)₂, CaSO₄,Ca(CH₃COO)₂, CaCl₂, CaBr₂, Ca(NO₃)₂, Ca(ClO₄)₂, CaI₂, Ca(SCN)₂,Ba₃(PO₄)₂, Ba₂HPO₄, Ba(H₂PO₄)₂, BaSO₄, Ba(CH₃COO)₂, BaCl₂, BaBr₂,Ba(NO₃)₂, Ba(ClO₄)₂, BaI₂, and Ba(SCN)₂. Particularly preferred are NHacetate, MgCl₂, KH₂PO₄, Na₂SO₄, KCl, NaCl, and CaCl₂, such as, forexample, the chloride or acetate (trifluoroacetate) salts.

Generally, peptides and variants (at least those containing peptidelinkages between amino acid residues) may be synthesized by theFmoc-polyamide mode of solid-phase peptide synthesis as disclosed byLukas et al. (Lukas et al., 1981) and by references as cited therein.Temporary N-amino group protection is afforded by the9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of thishighly base-labile protecting group is done using 20% piperidine in N,N-dimethylformamide. Side-chain functionalities may be protected astheir butyl ethers (in the case of serine threonine and tyrosine), butylesters (in the case of glutamic acid and aspartic acid),butyloxycarbonyl derivative (in the case of lysine and histidine),trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversedN,N-dicyclohexylcarbodiimide/1hydroxybenzotriazole mediated couplingprocedure. All coupling and deprotection reactions are monitored usingninhydrin, trinitrobenzene sulphonic acid or isotin test procedures.Upon completion of synthesis, peptides are cleaved from the resinsupport with concomitant removal of side-chain protecting groups bytreatment with 95% trifluoroacetic acid containing a 50% scavenger mix.Scavengers commonly used include ethanedithiol, phenol, anisole andwater, the exact choice depending on the constituent amino acids of thepeptide being synthesized. Also a combination of solid phase andsolution phase methodologies for the synthesis of peptides is possible(see, for example, (Bruckdorfer et al., 2004), and the references ascited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of,techniques such as recrystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitrile/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

For the identification of peptides of the present invention, twodatabases of RNA expression data were compared together: RNASeq tumordata generated by the TCGA Research Network(http://cancergenome.nih.gov/) and RNASeq data (GTEx) covering around3000 normal (healthy) tissue samples (Lonsdale, 2013). Genes werescreened, with were over-expressed in tumor tissues samples comparedwith the normal (healthy) tissue samples. Then, cancer-associatedpeptides derived from the protein products of these genes wereidentified by mass spectrometry using the XPRESIDENT™ platform asdescribed herein.

In order to select over-presented peptides, a presentation profile iscalculated showing the median sample presentation as well as replicatevariation. The profile juxtaposes samples of the tumor entity ofinterest to a baseline of normal tissue samples. Each of these profilescan then be consolidated into an over-presentation score by calculatingthe p-value of a Linear Mixed-Effects Model (Pinheiro et al., 2015)adjusting for multiple testing by False Discovery Rate (Benjamini andHochberg, 1995) (cf. Example 1, FIG. 1).

For the identification and relative quantitation of HLA ligands by massspectrometry, HLA molecules from shock-frozen tissue samples werepurified and HLA-associated peptides were isolated. The isolatedpeptides were separated and sequences were identified by onlinenano-electrospray-ionization (nanoESl) liquid chromatography-massspectrometry (LC-MS) experiments. The resulting peptide sequences wereverified by comparison of the fragmentation pattern of naturaltumor-associated peptides (TUMAPs) recorded from cancer samples (N=450A*02-positive samples, N=211 A*24-positive samples) with thefragmentation patterns of corresponding synthetic reference peptides ofidentical sequences. Since the peptides were directly identified asligands of HLA molecules of primary tumors, these results provide directevidence for the natural processing and presentation of the identifiedpeptides on primary cancer tissue obtained from A*02 and/orA*24-positive cancer patients.

The discovery pipeline XPRESIDENT® v2.1 (see, for example, US2013-0096016, which is hereby incorporated by reference in its entirety)allows the identification and selection of relevant over-presentedpeptide vaccine candidates based on direct relative quantitation ofHLA-restricted peptide levels on cancer tissues in comparison to severaldifferent non-cancerous tissues and organs. This was achieved by thedevelopment of label-free differential quantitation using the acquiredLC-MS data processed by a proprietary data analysis pipeline, combiningalgorithms for sequence identification, spectral clustering, ioncounting, retention time alignment, charge state deconvolution andnormalization.

Presentation levels including error estimates for each peptide andsample were established. Peptides exclusively presented on tumor tissueand peptides over-presented in tumor versus non-cancerous tissues andorgans have been identified.

HLA-peptide complexes from tissue samples were purified andHLA-associated peptides were isolated and analyzed by LC-MS (seeexamples). All TUMAPs contained in the present application wereidentified with this approach on primary cancer samples confirming theirpresentation on primary glioblastoma, breast cancer, colorectal cancer,renal cell carcinoma, chronic lymphocytic leukemia, hepatocellularcarcinoma, non-small cell and small cell lung cancer, Non-Hodgkinlymphoma, acute myeloid leukemia, ovarian cancer, pancreatic cancer,prostate cancer, esophageal cancer including cancer of thegastric-esophageal junction, gallbladder cancer and cholangiocarcinoma,melanoma, gastric cancer, urinary bladder cancer, or uterine cancer.

TUMAPs identified on multiple cancer and normal tissues were quantifiedusing ion-counting of label-free LC-MS data. The method assumes thatLC-MS signal areas of a peptide correlate with its abundance in thesample. All quantitative signals of a peptide in various LC-MSexperiments were normalized based on central tendency, averaged persample and merged into a bar plot, called presentation profile. Thepresentation profile consolidates different analysis methods likeprotein database search, spectral clustering, charge state deconvolution(decharging) and retention time alignment and normalization.

Furthermore, the discovery pipeline XPRESIDENT® v2.1 allows the directabsolute quantitation of MHC-, preferably HLA-restricted, peptide levelson cancer or other infected tissues. Briefly, the total cell count wascalculated from the total DNA content of the analyzed tissue sample. Thetotal peptide amount for a TUMAP in a tissue sample was measured bynanoLC-MS/MS as the ratio of the natural TUMAP and a known amount of anisotope-labelled version of the TUMAP, the so-called internal standard.The efficiency of TUMAP isolation was determined by spiking peptide:MHCcomplexes of all selected TUMAPs into the tissue lysate at the earliestpossible point of the TUMAP isolation procedure and their detection bynanoLC-MS/MS following completion of the peptide isolation procedure.The total cell count and the amount of total peptide were calculatedfrom triplicate measurements per tissue sample. The peptide-specificisolation efficiencies were calculated as an average from 10 spikeexperiments each measured as a triplicate (see Example 6 and Table 22)

This combined analysis of RNA expression and mass spectrometry dataresulted in the 289 peptides of the present invention.

Besides over-presentation of the peptide, mRNA expression of theunderlying gene was tested. mRNA data were obtained via RNASeq analysesof normal tissues and cancer tissues (cf. Example 2, FIG. 2). Anadditional source of normal tissue data was a database of publiclyavailable RNA expression data from around 3000 normal tissue samples(Lonsdale, 2013). Peptides which are derived from proteins whose codingmRNA is highly expressed in cancer tissue, but very low or absent invital normal tissues, were preferably included in the present invention.

The present invention provides peptides that are useful in treatingcancers/tumors, preferably glioblastoma, breast cancer, colorectalcancer, renal cell carcinoma, chronic lymphocytic leukemia,hepatocellular carcinoma, non-small cell and small cell lung cancer,Non-Hodgkin lymphoma, acute myeloid leukemia, ovarian cancer, pancreaticcancer, prostate cancer, esophageal cancer including cancer of thegastric-esophageal junction, gallbladder cancer and cholangiocarcinoma,melanoma, gastric cancer, urinary bladder cancer, head and neck squamouscell carcinoma, and uterine cancer that over- or exclusively present thepeptides of the invention. These peptides were shown by massspectrometry to be naturally presented by HLA molecules on primary humancancer samples.

Many of the source gene/proteins (also designated “full-length proteins”or “underlying proteins”) from which the peptides are derived were shownto be highly over-expressed in cancer compared with normaltissues—“normal tissues” in relation to this invention shall mean eitherhealthy cells or tissue derived from the same organ as the tumor, orother normal tissue cells, demonstrating a high degree of tumorassociation of the source genes (see Example 2). Moreover, the peptidesthemselves are strongly over-presented on tumor tissue—“tumor tissue” inrelation to this invention shall mean a sample from a patient sufferingfrom cancer, but not on normal tissues (see Example 1).

HLA-bound peptides can be recognized by the immune system, specificallyT lymphocytes. T cells can destroy the cells presenting the recognizedHLA/peptide complex, e.g. glioblastoma, breast cancer, colorectalcancer, renal cell carcinoma, chronic lymphocytic leukemia,hepatocellular carcinoma, non-small cell and small cell lung cancer,Non-Hodgkin lymphoma, acute myeloid leukemia, ovarian cancer, pancreaticcancer, prostate cancer, esophageal cancer including cancer of thegastric-esophageal junction, gallbladder cancer and cholangiocarcinoma,melanoma, gastric cancer, urinary bladder cancer, or uterine cancercells presenting the derived peptides.

The peptides of the present invention have been shown to be capable ofstimulating T cell responses and/or are over-presented and thus can beused for the production of antibodies and/or TCRs, such as soluble TCRs,according to the present invention (see Example 3). Furthermore, thepeptides when complexed with the respective MHC can be used for theproduction of antibodies and/or TCRs, in particular sTCRs, according tothe present invention, as well. Respective methods are well known to theperson of skill, and can be found in the respective literature as well(see also below). Thus, the peptides of the present invention are usefulfor generating an immune response in a patient by which tumor cells canbe destroyed. An immune response in a patient can be induced by directadministration of the described peptides or suitable precursorsubstances (e.g. elongated peptides, proteins, or nucleic acids encodingthese peptides) to the patient, ideally in combination with an agentenhancing the immunogenicity (i.e. an adjuvant). The immune responseoriginating from such a therapeutic vaccination can be expected to behighly specific against tumor cells because the target peptides of thepresent invention are not presented on normal tissues in comparable copynumbers, preventing the risk of undesired autoimmune reactions againstnormal cells in the patient. In this context, particularly preferred arethe peptides of the invention selected from the group consisting of SEQID NO: 2, 24, 32, 39, 64, 72, 106, 149, 251, 305, and 306.

The present description further relates to T-cell receptors (TCRs)comprising an alpha chain and a beta chain (“alpha/beta TCRs”). Alsoprovided are peptides according to the invention capable of binding toTCRs and antibodies when presented by an MHC molecule. The presentdescription also relates to nucleic acids, vectors and host cells forexpressing TCRs and peptides of the present description; and methods ofusing the same. Again, particularly preferred in this context are thepeptides of the invention selected from the group consisting of SEQ IDNO: 2, 24, 32, 39, 64, 72, 106, 149, 251, 305, and 306.

The term “T-cell receptor” (abbreviated TCR) refers to a heterodimericmolecule comprising an alpha polypeptide chain (alpha chain) and a betapolypeptide chain (beta chain), wherein the heterodimeric receptor iscapable of binding to a peptide antigen presented by an HLA molecule.The term also includes so-called gamma/delta TCRs.

In one embodiment the description provides a method of producing a TCRas described herein, the method comprising culturing a host cell capableof expressing the TCR under conditions suitable to promote expression ofthe TCR.

The description in another aspect relates to methods according to thedescription, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell or the antigen is loadedonto class I or II MHC tetramers by tetramerizing the antigen/class I orII MHC complex monomers.

The alpha and beta chains of alpha/beta TCR's, and the gamma and deltachains of gamma/delta TCRs, are generally regarded as each having two“domains”, namely variable and constant domains. The variable domainconsists of a concatenation of variable region (V), and joining region(J). The variable domain may also include a leader region (L). Beta anddelta chains may also include a diversity region (D). The alpha and betaconstant domains may also include C-terminal transmembrane (TM) domainsthat anchor the alpha and beta chains to the cell membrane.

With respect to gamma/delta TCRs, the term “TCR gamma variable domain”as used herein refers to the concatenation of the TCR gamma V (TRGV)region without leader region (L), and the TCR gamma J (TRGJ) region, andthe term TCR gamma constant domain refers to the extracellular TRGCregion, or to a C-terminal truncated TRGC sequence. Likewise the term“TCR delta variable domain” refers to the concatenation of the TCR deltaV (TRDV) region without leader region (L) and the TCR delta D/J(TRDD/TRDJ) region, and the term “TCR delta constant domain” refers tothe extracellular TRDC region, or to a C-terminal truncated TRDCsequence.

TCRs of the present description preferably bind to an peptide-HLAmolecule complex with a binding affinity (KD) of about 100 μM or less,about 50 μM or less, about 25 μM or less, or about 10 μM or less. Morepreferred are high affinity TCRs having binding affinities of about 1 μMor less, about 100 nM or less, about 50 nM or less, about 25 nM or less.Non-limiting examples of preferred binding affinity ranges for TCRs ofthe present invention include about 1 nM to about 10 nM; about 10 nM toabout 20 nM; about 20 nM to about 30 nM; about 30 nM to about 40 nM;about 40 nM to about 50 nM; about 50 nM to about 60 nM; about 60 nM toabout 70 nM; about 70 nM to about 80 nM; about 80 nM to about 90 nM; andabout 90 nM to about 100 nM.

As used herein in connect with TCRs of the present description,“specific binding” and grammatical variants thereof are used to mean aTCR having a binding affinity (KD) for a peptide-HLA molecule complex of100 μM or less.

Alpha/beta heterodimeric TCRs of the present description may have anintroduced disulfide bond between their constant domains. Preferred TCRsof this type include those which have a TRAC constant domain sequenceand a TRBC1 or TRBC2 constant domain sequence except that Thr 48 of TRACand Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, the saidcysteines forming a disulfide bond between the TRAC constant domainsequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

With or without the introduced inter-chain bond mentioned above,alpha/beta hetero-dimeric TCRs of the present description may have aTRAC constant domain sequence and a TRBC1 or TRBC2 constant domainsequence, and the TRAC constant domain sequence and the TRBC1 or TRBC2constant domain sequence of the TCR may be linked by the nativedisulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 ofTRBC1 or TRBC2.

TCRs of the present description may comprise a detectable label selectedfrom the group consisting of a radionuclide, a fluorophore and biotin.TCRs of the present description may be conjugated to a therapeuticallyactive agent, such as a radionuclide, a chemotherapeutic agent, or atoxin.

In an embodiment, a TCR of the present description having at least onemutation in the alpha chain and/or having at least one mutation in thebeta chain has modified glycosylation compared to the unmutated TCR.

In an embodiment, a TCR comprising at least one mutation in the TCRalpha chain and/or TCR beta chain has a binding affinity for, and/or abinding half-life for, a peptide-HLA molecule complex, which is at leastdouble that of a TCR comprising the unmutated TCR alpha chain and/orunmutated TCR beta chain. Affinity-enhancement of tumor-specific TCRs,and its exploitation, relies on the existence of a window for optimalTCR affinities. The existence of such a window is based on observationsthat TCRs specific for HLA-A2-restricted pathogens have KD values thatare generally about 10-fold lower when compared to TCRs specific forHLA-A2-restricted tumor-associated self-antigens. It is now known,although tumor antigens have the potential to be immunogenic, becausetumors arise from the individual's own cells only mutated proteins orproteins with altered translational processing will be seen as foreignby the immune system. Antigens that are up-regulated or overexpressed(so called self-antigens) will not necessarily induce a functionalimmune response against the tumor: T-cells expressing TCRs that arehighly reactive to these antigens will have been negatively selectedwithin the thymus in a process known as central tolerance, meaning thatonly T-cells with low-affinity TCRs for self-antigens remain. Therefore,affinity of TCRs or variants of the present description to pepides canbe enhanced by methods well known in the art.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising incubating PBMCs from HLA-A*02-negative healthy donors withA2/peptide monomers, incubating the PBMCs with tetramer-phycoerythrin(PE) and isolating the high avidity T-cells by fluo-rescence activatedcell sorting (FACS)—Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising obtaining a transgenic mouse with the entire human TCRap geneloci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCRrepertoire that compensates for mouse TCR deficiency, immunizing themouse with a peptide, incubating PBMCs obtained from the transgenic micewith tetramer-phycoerythrin (PE), and isolating the high avidity T-cellsby fluorescence activated cell sorting (FACS)—Calibur analysis.

In one aspect, to obtain T-cells expressing TCRs of the presentdescription, nucleic acids encoding TCR-alpha and/or TCR-beta chains ofthe present description are cloned into expression vectors, such asgamma retrovirus or lentivirus. The recombinant viruses are generatedand then tested for functionality, such as antigen specificity andfunctional avidity. An aliquot of the final product is then used totransduce the target T-cell population (generally purified from patientPBMCs), which is expanded before infusion into the patient.

In another aspect, to obtain T-cells expressing TCRs of the presentdescription, TCR RNAs are synthesized by techniques known in the art,e.g., in vitro transcription sys-tems. The in vitro-synthesized TCR RNAsare then introduced into primary CD8+ T-cells obtained from healthydonors by electroporation to re-express tumor specific TCR-alpha and/orTCR-beta chains.

To increase the expression, nucleic acids encoding TCRs of the presentdescription may be operably linked to strong promoters, such asretroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murinestem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin,ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter,elongation factor (EF)-1a and the spleen focus-forming virus (SFFV)promoter. In a preferred embodiment, the promoter is heterologous to thenucleic acid being expressed.

In addition to strong promoters, TCR expression cassettes of the presentdescription may contain additional elements that can enhance transgeneexpression, including a central polypurine tract (cPPT), which promotesthe nuclear translocation of lentiviral constructs (Follenzi et al.,2000), and the woodchuck hepatitis virus posttranscriptional regulatoryelement (wPRE), which increases the level of transgene expression byincreasing RNA stability (Zufferey et al., 1999).

The alpha and beta chains of a TCR of the present invention may beencoded by nucleic acids located in separate vectors, or may be encodedby polynucleotides located in the same vector.

Achieving high-level TCR surface expression requires that both theTCR-alpha and TCR-beta chains of the introduced TCR be transcribed athigh levels. To do so, the TCR-alpha and TCR-beta chains of the presentdescription may be cloned into bi-cistronic constructs in a singlevector, which has been shown to be capable of over-coming this obstacle.The use of a viral intraribosomal entry site (IRES) between theTCR-alpha and TCR-beta chains results in the coordinated expression ofboth chains, because the TCR-alpha and TCR-beta chains are generatedfrom a single transcript that is broken into two proteins duringtranslation, ensuring that an equal molar ratio of TCR-alpha andTCR-beta chains are produced (Schmitt et al., 2009).

Nucleic acids encoding TCRs of the present description may be codonoptimized to increase expression from a host cell. Redundancy in thegenetic code allows some amino acids to be encoded by more than onecodon, but certain codons are less “optimal” than others because of therelative availability of matching tRNAs as well as other factors(Gustafsson et al., 2004). Modifying the TCR-alpha and TCR-beta genesequences such that each amino acid is encoded by the optimal codon formammalian gene expression, as well as eliminating mRNA instabilitymotifs or cryptic splice sites, has been shown to significantly enhanceTCR-alpha and TCR-beta gene expression (Scholten et al., 2006).

Furthermore, mispairing between the introduced and endogenous TCR chainsmay result in the acquisition of specificities that pose a significantrisk for autoimmunity. For example, the formation of mixed TCR dimersmay reduce the number of CD3 molecules available to form properly pairedTCR complexes, and therefore can significantly decrease the functionalavidity of the cells expressing the introduced TCR (Kuball et al.,2007).

To reduce mispairing, the C-terminus domain of the introduced TCR chainsof the present description may be modified in order to promoteinterchain affinity, while de-creasing the ability of the introducedchains to pair with the endogenous TCR. These strategies may includereplacing the human TCR-alpha and TCR-beta C-terminus domains with theirmurine counterparts (murinized C-terminus domain); generating a secondinterchain disulfide bond in the C-terminus domain by introducing asecond cysteine residue into both the TCR-alpha and TCR-beta chains ofthe introduced TCR (cysteine modification); swapping interactingresidues in the TCR-alpha and TCR-beta chain C-terminus domains(“knob-in-hole”); and fusing the variable domains of the TCR-alpha andTCR-beta chains directly to CD3ζ (CD3ζ fusion) (Schmitt et al., 2009).

In an embodiment, a host cell is engineered to express a TCR of thepresent description. In preferred embodiments, the host cell is a humanT-cell or T-cell progenitor. In some embodiments the T-cell or T-cellprogenitor is obtained from a cancer patient. In other embodiments theT-cell or T-cell progenitor is obtained from a healthy donor. Host cellsof the present description can be allogeneic or autologous with respectto a patient to be treated. In one embodiment, the host is a gamma/deltaT-cell transformed to express an alpha/beta TCR.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt (see alsoabove). As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH2 group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methane sulfonic acid, ethane sulfonic acid, p-toluenesulfonicacid, salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on a peptide are prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates), trifluoroacetates or hydrochloric acid (chlorides).

Preferably, the medicament of the present invention is animmunotherapeutic such as a vaccine. It may be administered directlyinto the patient, into the affected organ or systemically i.d., i.m.,s.c., i.p. and i.v., or applied ex vivo to cells derived from thepatient or a human cell line which are subsequently administered to thepatient, or used in vitro to select a subpopulation of immune cellsderived from the patient, which are then re-administered to the patient.If the nucleic acid is administered to cells in vitro, it may be usefulfor the cells to be transfected so as to co-express immune-stimulatingcytokines, such as interleukin-2. The peptide may be substantially pure,or combined with an immune-stimulating adjuvant (see below) or used incombination with immune-stimulatory cytokines, or be administered with asuitable delivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). Thepeptide may also be tagged, may be a fusion protein, or may be a hybridmolecule. The peptides whose sequence is given in the present inventionare expected to stimulate CD4 or CD8 T cells. However, stimulation ofCD8 T cells is more efficient in the presence of help provided by CD4T-helper cells. Thus, for MHC Class I epitopes that stimulate CD8 Tcells the fusion partner or sections of a hybrid molecule suitablyprovide epitopes which stimulate CD4-positive T cells. CD4- andCD8-stimulating epitopes are well known in the art and include thoseidentified in the present invention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 289, 305, and306, and at least one additional peptide, preferably two to 50, morepreferably two to 25, even more preferably two to 20 and most preferablytwo, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, seventeen or eighteen peptides.The peptide(s) may be derived from one or more specific TAAs and maybind to MHC class I molecules.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by Saiki RK, et al. (Saiki et al., 1988). This method may be used for introducingthe DNA into a suitable vector, for example by engineering in suitablerestriction sites, or it may be used to modify the DNA in other usefulways as is known in the art. If viral vectors are used, pox- oradenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (Ylps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).CMV promoter-based vectors (for example from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG,3×FLAG, c-myc or MAT. These fusion proteins allow for detection,purification and analysis of recombinant protein. Dual-tagged fusionsprovide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the f1 origin. Vectorscontaining the pre-pro-trypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of theinvention are encoded and thus expressed in a successive order (similarto “beads on a string” constructs). In doing so, the peptides or peptidevariants may be linked or fused together by stretches of linker aminoacids, such as for example LLLLLL, or may be linked without anyadditional peptide(s) between them. These constructs can also be usedfor cancer therapy, and may induce immune responses both involving MHC Iand MHC II.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells which are human embryonic kidney cells. Preferred insect cells areSf9 cells which can be transfected with baculovirus expression vectors.An overview regarding the choice of suitable host cells for expressioncan be found in, for example, the textbook of Paulina Balbás and ArgeliaLorence “Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,” Part One, Second Edition, ISBN978-1-58829-262-9, and other literature known to the person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (Cohen et al.,1972) and (Green and Sambrook, 2012). Transformation of yeast cells isdescribed in Sherman et al. (Sherman et al., 1986). The method of Beggs(Beggs, 1978) is also useful. With regard to vertebrate cells, reagentsuseful in transfecting such cells, for example calcium phosphate andDEAE-dextran or liposome formulations, are available from StratageneCloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877,USA. Electroporation is also useful for transforming and/or transfectingcells and is well known in the art for transforming yeast cell,bacterial cells, insect cells and vertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantiallypure, or contained in a suitable vector or delivery system. The nucleicacid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods fordesigning and introducing such a nucleic acid are well known in the art.An overview is provided by e.g. Teufel et al. (Teufel et al., 2005).Polynucleotide vaccines are easy to prepare, but the mode of action ofthese vectors in inducing an immune response is not fully understood.Suitable vectors and delivery systems include viral DNA and/or RNA, suchas systems based on adenovirus, vaccinia virus, retroviruses, herpesvirus, adeno-associated virus or hybrids containing elements of morethan one virus. Non-viral delivery systems include cationic lipids andcationic polymers and are well known in the art of DNA delivery.Physical delivery, such as via a “gene-gun” may also be used. Thepeptide or peptides encoded by the nucleic acid may be a fusion protein,for example with an epitope that stimulates T cells for the respectiveopposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CD8-positive T cellsand helper-T (TH) cells to an antigen, and would thus be considereduseful in the medicament of the present invention. Suitable adjuvantsinclude, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®,AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligandsderived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod(ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13,IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, ISCOMATRIX, ISCOMs, Juvlmmune®, LipoVac, MALP2, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system,poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995). Also cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-), accelerating the maturation of dendritic cellsinto efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF,IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporatedherein by reference in its entirety) and acting as immunoadjuvants(e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich etal., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonal®, poly-(ICLC),poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, Bevacizumab®, celebrex, NCX-4016, sildenafil, tadalafil,vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632,pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodiestargeting key structures of the immune system (e.g. anti-CD40,anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which may acttherapeutically and/or as an adjuvant. The amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan without undueexperimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpGoligonucleotides and derivates, poly-(I:C) and derivates, RNA,sildenafil, and particulate formulations with PLG or virosomes.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonal®) and anti-CD40 mAB, or combinationsthereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavors, lubricants, etc. Thepeptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). Thecomposition can be used for a prevention, prophylaxis and/or therapy ofadenomatous or cancerous diseases. Exemplary formulations can be foundin, for example, EP2112253.

It is important to realize that the immune response triggered by thevaccine according to the invention attacks the cancer in differentcell-stages and different stages of development. Furthermore differentcancer associated signaling pathways are attacked. This is an advantageover vaccines that address only one or few targets, which may cause thetumor to easily adapt to the attack (tumor escape). Furthermore, not allindividual tumors express the same pattern of antigens. Therefore, acombination of several tumor-associated peptides ensures that everysingle tumor bears at least some of the targets. The composition isdesigned in such a way that each tumor is expected to express several ofthe antigens and cover several independent pathways necessary for tumorgrowth and maintenance. Thus, the vaccine can easily be used“off-the-shelf” for a larger patient population. This means that apre-selection of patients to be treated with the vaccine can berestricted to HLA typing, does not require any additional biomarkerassessments for antigen expression, but it is still ensured that severaltargets are simultaneously attacked by the induced immune response,which is important for efficacy (Banchereau et al., 2001; Walter et al.,2012).

As used herein, the term “scaffold” refers to a molecule thatspecifically binds to an (e.g. antigenic) determinant. In oneembodiment, a scaffold is able to direct the entity to which it isattached (e.g. a (second) antigen binding moiety) to a target site, forexample to a specific type of tumor cell or tumor stroma bearing theantigenic determinant (e.g. the complex of a peptide with MHC, accordingto the application at hand). In another embodiment a scaffold is able toactivate signaling through its target antigen, for example a T cellreceptor complex antigen. Scaffolds include but are not limited toantibodies and fragments thereof, antigen binding domains of anantibody, comprising an antibody heavy chain variable region and anantibody light chain variable region, binding proteins comprising atleast one ankyrin repeat motif and single domain antigen binding (SDAB)molecules, aptamers, (soluble) TCRs and (modified) cells such asallogenic or autologous T cells. To assess whether a molecule is ascaffold binding to a target, binding assays can be performed.

“Specific” binding means that the scaffold binds the peptide-MHC-complexof interest better than other naturally occurring peptide-MHC-complexes,to an extent that a scaffold armed with an active molecule that is ableto kill a cell bearing the specific target is not able to kill anothercell without the specific target but presenting other peptide-MHCcomplex(es). Binding to other peptide-MHC complexes is irrelevant if thepeptide of the cross-reactive peptide-MHC is not naturally occurring,i.e. not derived from the human HLA-peptidome. Tests to assess targetcell killing are well known in the art. They should be performed usingtarget cells (primary cells or cell lines) with unaltered peptide-MHCpresentation, or cells loaded with peptides such that naturallyoccurring peptide-MHC levels are reached.

Each scaffold can comprise a labelling which provides that the boundscaffold can be detected by determining the presence or absence of asignal provided by the label. For example, the scaffold can be labelledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example afluorescence-labelling, for example provided by a fluorescence dye, canprovide a visualization of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry.

Each scaffold can be conjugated with a second active molecule such asfor example IL-21, anti-CD3, and anti-CD28.

For further information on polypeptide scaffolds see for example thebackground section of WO 2014/071978A1 and the references cited therein.

The present invention further relates to aptamers. Aptamers (see forexample WO 2014/191359 and the literature as cited therein) are shortsingle-stranded nucleic acid molecules, which can fold into definedthree-dimensional structures and recognize specific target structures.They have appeared to be suitable alternatives for developing targetedtherapies. Aptamers have been shown to selectively bind to a variety ofcomplex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identifiedwithin the past decade and provide means for developing diagnostic andtherapeutic approaches. Since aptamers have been shown to possess almostno toxicity and immunogenicity they are promising candidates forbiomedical applications. Indeed aptamers, for example prostate-specificmembrane-antigen recognizing aptamers, have been successfully employedfor targeted therapies and shown to be functional in xenograft in vivomodels. Furthermore, aptamers recognizing specific tumor cell lines havebeen identified.

DNA aptamers can be selected to reveal broad-spectrum recognitionproperties for various cancer cells, and particularly those derived fromsolid tumors, while non-tumorigenic and primary healthy cells are notrecognized. If the identified aptamers recognize not only a specifictumor sub-type but rather interact with a series of tumors, this rendersthe aptamers applicable as so-called broad-spectrum diagnostics andtherapeutics.

Further, investigation of cell-binding behavior with flow cytometryshowed that the aptamers revealed very good apparent affinities that arewithin the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. Further, itcould be shown that some of the aptamers are taken up by tumor cells andthus can function as molecular vehicles for the targeted delivery ofanti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex targets such as cells andtissues and complexes of the peptides comprising, preferably consistingof, a sequence according to any of SEQ ID NO 1 to SEQ ID NO 289, 305,and 306, according to the invention at hand with the MHC molecule, usingthe cell-SELEX (Systematic Evolution of Ligands by Exponentialenrichment) technique.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen (preferably a peptide according to thepresent invention), the method comprising: immunizing a geneticallyengineered non-human mammal comprising cells expressing said human majorhistocompatibility complex (MHC) class I or II with a soluble form of aMHC class I or II molecule being complexed with said HLA-restrictedantigen; isolating mRNA molecules from antibody producing cells of saidnon-human mammal; producing a phage display library displaying proteinmolecules encoded by said mRNA molecules; and isolating at least onephage from said phage display library, said at least one phagedisplaying said antibody specifically binding to said human majorhistocompatibility complex (MHC) class I or II being complexed with saidHLA-restricted antigen.

It is thus a further aspect of the invention to provide an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with a HLA-restricted antigen, wherein theantibody preferably is a polyclonal antibody, monoclonal antibody,bi-specific antibody and/or a chimeric antibody.

Respective methods for producing such antibodies and single chain classI major histocompatibility complexes, as well as other tools for theproduction of these antibodies are disclosed in WO 03/068201, WO2004/084798, WO 01/72768, WO 03/070752, and in publications (Cohen etal., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), which for thepurposes of the present invention are all explicitly incorporated byreference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isalso regarded as “specific” in the context of the present invention.

The present invention relates to a peptide comprising a sequence that isselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 289,305, and 306, or a variant thereof which is at least 88% homologous(preferably identical) to SEQ ID NO: 1 to SEQ ID NO: 289, 305, and 306or a variant thereof that induces T cells cross-reacting with saidpeptide, wherein said peptide is not the underlying full-lengthpolypeptide.

The present invention further relates to a peptide comprising a sequencethat is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:289, 305, and 306 or a variant thereof which is at least 88% homologous(preferably identical) to SEQ ID NO: 1 to SEQ ID NO: 289, 305, and 306,wherein said peptide or variant has an overall length of between 8 and100, preferably between 8 and 30, and most preferred between 8 and 14amino acids.

The present invention further relates to the peptides according to theinvention that have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II.

The present invention further relates to the peptides according to theinvention wherein the peptide consists or consists essentially of anamino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 289, 305,and 306.

The present invention further relates to the peptides according to theinvention, wherein the peptide is (chemically) modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to theinvention, wherein the peptide is part of a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or wherein the peptide is fusedto (or into) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the invention, provided that the peptide is notthe complete (full) human protein.

The present invention further relates to the nucleic acid according tothe invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing a nucleic acid according to the present invention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine, in particular in the treatment of cancers such asglioblastoma, breast cancer, colorectal cancer, renal cell carcinoma,chronic lymphocytic leukemia, hepatocellular carcinoma, non-small celland small cell lung cancer, Non-Hodgkin lymphoma, acute myeloidleukemia, ovarian cancer, pancreatic cancer, prostate cancer, esophagealcancer including cancer of the gastric-esophageal junction, gallbladdercancer and cholangiocarcinoma, melanoma, gastric cancer, urinary bladdercancer, head and neck squamous cell carcinoma, or uterine cancer.

The present invention further relates to a host cell comprising anucleic acid according to the invention or an expression vectoraccording to the invention.

The present invention further relates to the host cell according to thepresent invention that is an antigen presenting cell, and preferably adendritic cell.

The present invention further relates to a method of producing a peptideaccording to the present invention, said method comprising culturing thehost cell according to the present invention, and isolating the peptidefrom said host cell or its culture medium.

The present invention further relates to the method according to thepresent invention, where-in the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellby contacting a sufficient amount of the antigen with anantigen-presenting cell.

The present invention further relates to the method according to theinvention, wherein the antigen-presenting cell comprises an expressionvector capable of expressing said peptide containing SEQ ID NO: 1 to SEQID NO: 289, 305, and 306 or said variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellsselectively recognizes a cell which aberrantly expresses a polypeptidecomprising an amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as according to the present invention.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated cytotoxic T lymphocyteaccording to the present invention as a medicament or in the manufactureof a medicament. The present invention further relates to a useaccording to the present invention, wherein the medicament is activeagainst cancer.

The present invention further relates to a use according to theinvention, wherein the medicament is a vaccine. The present inventionfurther relates to a use according to the invention, wherein themedicament is active against cancer.

The present invention further relates to a use according to theinvention, wherein said cancer cells are cells or other solid orhaematological tumor cells such as glioblastoma, breast cancer,colorectal cancer, renal cell carcinoma, chronic lymphocytic leukemia,hepatocellular carcinoma, non-small cell and small cell lung cancer,Non-Hodgkin lymphoma, acute myeloid leukemia, ovarian cancer, pancreaticcancer, prostate cancer, esophageal cancer including cancer of thegastric-esophageal junction, gallbladder cancer and cholangiocarcinoma,melanoma, gastric cancer, urinary bladder cancer, head and neck squamouscell carcinoma, or uterine cancer cells.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention,herein called “targets” that can be used in the diagnosis and/orprognosis of glioblastoma, breast cancer, colorectal cancer, renal cellcarcinoma, chronic lymphocytic leukemia, hepatocellular carcinoma,non-small cell and small cell lung cancer, Non-Hodgkin lymphoma, acutemyeloid leukemia, ovarian cancer, pancreatic cancer, prostate cancer,esophageal cancer including cancer of the gastric-esophageal junction,gallbladder cancer and cholangiocarcinoma, melanoma, gastric cancer,urinary bladder cancer, head and neck squamous cell carcinoma, oruterine cancer. The present invention also relates to the use of thesenovel targets for cancer treatment.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments (e.g. CDRs, Fv, Fab and Fc fragments) orpolymers of those immunoglobulin molecules and humanized versions ofimmunoglobulin molecules, as long as they exhibit any of the desiredproperties (e.g., specific binding of a glioblastoma, breast cancer,colorectal cancer, renal cell carcinoma, chronic lymphocytic leukemia,hepatocellular carcinoma, non-small cell and small cell lung cancer,Non-Hodgkin lymphoma, acute myeloid leukemia, ovarian cancer, pancreaticcancer, prostate cancer, esophageal cancer including cancer of thegastric-esophageal junction, gallbladder cancer and cholangiocarcinoma,melanoma, gastric cancer, urinary bladder cancer, head and neck squamouscell carcinoma, or uterine cancer marker (poly)peptide, delivery of atoxin to a cancer cell expressing a cancer marker gene at an increasedlevel, and/or inhibiting the activity of a cancer marker polypeptide)according to the invention.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length glioblastoma, breast cancer, colorectal cancer,renal cell carcinoma, chronic lymphocytic leukemia, hepatocellularcarcinoma, non-small cell and small cell lung cancer, Non-Hodgkinlymphoma, acute myeloid leukemia, ovarian cancer, pancreatic cancer,prostate cancer, esophageal cancer including cancer of thegastric-esophageal junction, gallbladder cancer and cholangiocarcinoma,melanoma, gastric cancer, urinary bladder cancer, head and neck squamouscell carcinoma, or uterine cancer marker polypeptides or fragmentsthereof may be used to generate the antibodies of the invention. Apolypeptide to be used for generating an antibody of the invention maybe partially or fully purified from a natural source, or may be producedusing recombinant DNA techniques.

For example, a cDNA encoding a peptide according to the presentinvention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO:289, 305, and 306 polypeptide, or a variant or fragment thereof, can beexpressed in prokaryotic cells (e.g., bacteria) or eukaryotic cells(e.g., yeast, insect, or mammalian cells), after which the recombinantprotein can be purified and used to generate a monoclonal or polyclonalantibody preparation that specifically bind the marker polypeptide forabove-mentioned cancers used to generate the antibody according to theinvention.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014)). Forexample, the antibodies may be tested in ELISA assays or, Western blots,immunohistochemical staining of formalin-fixed cancers or frozen tissuesections. After their initial in vitro characterization, antibodiesintended for therapeutic or in vivo diagnostic use are tested accordingto known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields a F(ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (μg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of an antibody, preferably for treating glioblastoma,breast cancer, colorectal cancer, renal cell carcinoma, chroniclymphocytic leukemia, hepatocellular carcinoma, non-small cell and smallcell lung cancer, Non-Hodgkin lymphoma, acute myeloid leukemia, ovariancancer, pancreatic cancer, prostate cancer, esophageal cancer includingcancer of the gastric-esophageal junction, gallbladder cancer andcholangiocarcinoma, melanoma, gastric cancer, urinary bladder cancer,head and neck squamous cell carcinoma, or uterine cancer, the efficacyof the therapeutic antibody can be assessed in various ways well knownto the skilled practitioner. For instance, the size, number, and/ordistribution of cancer in a subject receiving treatment may be monitoredusing standard tumor imaging techniques. A therapeutically-administeredantibody that arrests tumor growth, results in tumor shrinkage, and/orprevents the development of new tumors, compared to the disease coursethat would occurs in the absence of antibody administration, is anefficacious antibody for treatment of cancer.

It is a further aspect of the invention to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use asdrug, alpha and beta chain can be linked e.g. by non-native disulfidebonds, other covalent bonds (single-chain T-cell receptor), or bydimerization domains (Boulter et al., 2003; Card et al., 2004; Willcoxet al., 1999). The T-cell receptor can be linked to toxins, drugs,cytokines (see, for example, US 2013/0115191), and domains recruitingeffector cells such as an anti-CD3 domain, etc., in order to executeparticular functions on target cells. Moreover, it could be expressed inT cells used for adoptive transfer. Further information can be found inWO 2004/033685A1 and WO 2004/074322A1. A combination of sTCRs isdescribed in WO 2012/056407A1. Further methods for the production aredisclosed in WO 2013/057586A1.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present invention can be used to verify a pathologist'sdiagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moretargets of a protein selected from the group consisting of theabove-mentioned proteins, and the affinity value (Kd) is less than 1×10μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ.

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Ljunggren et al.(Ljunggren and Karre, 1985).

Preferably, before transfection the host cell expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class I molecules and of the co-stimulatormolecules are publicly available from the Gen Bank and EMBL databases.

In case of a MHC class I epitope being used as an antigen, the T cellsare CD8-positive T cells.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 289, 305, and 306, or avariant amino acid sequence thereof.

A number of other methods may be used for generating T cells in vitro.For example, autologous tumor-infiltrating lymphocytes can be used inthe generation of CTL. Plebanski et al. (Plebanski et al., 1995) madeuse of autologous peripheral blood lymphocytes (PLBs) in the preparationof T cells. Furthermore, the production of autologous T cells by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus is possible. Also, B cells can be used in theproduction of autologous T cells. In addition, macrophages pulsed withpeptide or polypeptide, or infected with recombinant virus, may be usedin the preparation of autologous T cells. S. Walter et al. (Walter etal., 2003) describe the in vitro priming of T cells by using artificialantigen presenting cells (aAPCs), which is also a suitable way forgenerating T cells against the peptide of choice. In the presentinvention, aAPCs were generated by the coupling of preformed MHC:peptidecomplexes to the surface of polystyrene particles (microbeads) bybiotin:streptavidin biochemistry. This system permits the exact controlof the MHC density on aAPCs, which allows to selectively elicit high- orlow-avidity antigen-specific T cell responses with high efficiency fromblood samples. Apart from MHC:peptide complexes, aAPCs should carryother proteins with co-stimulatory activity like anti-CD28 antibodiescoupled to their surface. Furthermore such aAPC-based systems oftenrequire the addition of appropriate soluble factors, e. g. cytokines,like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, andvaccinia-infected target cells. In addition plant viruses may be used(see, for example, Porta et al. (Porta et al., 1994) which describes thedevelopment of cowpea mosaic virus as a high-yielding system for thepresentation of foreign peptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 289,305, and 306.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease that can be readily tested for, anddetected.

In vivo, the target cells for the CD8-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class II; (Dengjel et al., 2006)).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to levels of expression in normal tissues orthat the gene is silent in the tissue from which the tumor is derivedbut in the tumor it is expressed. By “over-expressed” the inventors meanthat the polypeptide is present at a level at least 1.2-fold of thatpresent in normal tissue; preferably at least 2-fold, and morepreferably at least 5-fold or 10-fold the level present in normaltissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T cells are well knownin the art. Reviews can be found in: Gattioni et al. and Morgan et al.(Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the present invention includes the use of the peptidescomplexed with MHC to generate a T-cell receptor whose nucleic acid iscloned and is introduced into a host cell, preferably a T cell. Thisengineered T cell can then be transferred to a patient for therapy ofcancer.

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody,expression vector, cell, activated T cell, T-cell receptor or thenucleic acid encoding it, is useful for the treatment of disorders,characterized by cells escaping an immune response. Therefore anymolecule of the present invention may be used as medicament or in themanufacture of a medicament. The molecule may be used by itself orcombined with other molecule(s) of the invention or (a) knownmolecule(s).

The present invention is further directed at a kit comprising:

(a) a container containing a pharmaceutical composition as describedabove, in solution or in lyophilized form;

(b) optionally a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; and

(c) optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably, the administration is s.c., and most preferablyi.d. administration may be by infusion pump.

Since the peptides of the invention were isolated from glioblastoma,breast cancer, colorectal cancer, renal cell carcinoma, chroniclymphocytic leukemia, hepatocellular carcinoma, non-small cell and smallcell lung cancer, Non-Hodgkin lymphoma, acute myeloid leukemia, ovariancancer, pancreatic cancer, prostate cancer, esophageal cancer includingcancer of the gastric-esophageal junction, gallbladder cancer andcholangiocarcinoma, melanoma, gastric cancer, urinary bladder cancer, oruterine cancer, the medicament of the invention is preferably used totreat glioblastoma, breast cancer, colorectal cancer, renal cellcarcinoma, chronic lymphocytic leukemia, hepatocellular carcinoma,non-small cell and small cell lung cancer, Non-Hodgkin lymphoma, acutemyeloid leukemia, ovarian cancer, pancreatic cancer, prostate cancer,esophageal cancer including cancer of the gastric-esophageal junction,gallbladder cancer and cholangiocarcinoma, melanoma, gastric cancer,urinary bladder cancer, head and neck squamous cell carcinoma, oruterine cancer.

The present invention further relates to a method for producing apersonalized pharmaceutical for an individual patient comprisingmanufacturing a pharmaceutical composition comprising at least onepeptide selected from a warehouse of pre-screened TUMAPs, wherein the atleast one peptide used in the pharmaceutical composition is selected forsuitability in the individual patient. In one embodiment, thepharmaceutical composition is a vaccine. The method could also beadapted to produce T cell clones for down-stream applications, such asTCR isolations, or soluble antibodies, and other treatment options.

A “personalized pharmaceutical” shall mean specifically tailoredtherapies for one individual patient that will only be used for therapyin such individual patient, including actively personalized cancervaccines and adoptive cellular therapies using autologous patienttissue.

As used herein, the term “warehouse” shall refer to a group or set ofpeptides that have been pre-screened for immunogenicity and/orover-presentation in a particular tumor type. The term “warehouse” isnot intended to imply that the particular peptides included in thevaccine have been pre-manufactured and stored in a physical facility,although that possibility is contemplated. It is expressly contemplatedthat the peptides may be manufactured de novo for each individualizedvaccine produced, or may be pre-manufactured and stored. The warehouse(e.g. in the form of a database) is composed of tumor-associatedpeptides which were highly overexpressed in the tumor tissue ofglioblastoma, breast cancer, colorectal cancer, renal cell carcinoma,chronic lymphocytic leukemia, hepatocellular carcinoma, non-small celland small cell lung cancer, Non-Hodgkin lymphoma, acute myeloidleukemia, ovarian cancer, pancreatic cancer, prostate cancer, esophagealcancer including cancer of the gastric-esophageal junction, gallbladdercancer and cholangiocarcinoma, melanoma, gastric cancer, urinary bladdercancer, head and neck squamous cell carcinoma, or uterine cancerpatients with various HLA-A HLA-B and HLA-C alleles. It may contain MHCclass I and MHC class II peptides or elongated MHC class I peptides. Inaddition to the tumor associated peptides collected from several cancertissues, the warehouse may contain HLA-A*02 and HLA-A*24 markerpeptides. These peptides allow comparison of the magnitude of T-cellimmunity induced by TUMAPS in a quantitative manner and hence allowimportant conclusion to be drawn on the capacity of the vaccine toelicit anti-tumor responses. Secondly, they function as importantpositive control peptides derived from a “non-self” antigen in the casethat any vaccine-induced T-cell responses to TUMAPs derived from “self”antigens in a patient are not observed. And thirdly, it may allowconclusions to be drawn, regarding the status of immunocompetence of thepatient.

TUMAPs for the warehouse are identified by using an integratedfunctional genomics approach combining gene expression analysis, massspectrometry, and T-cell immunology (XPresident®). The approach assuresthat only TUMAPs truly present on a high percentage of tumors but not oronly minimally expressed on normal tissue, are chosen for furtheranalysis. For initial peptide selection, glioblastoma, breast cancer,colorectal cancer, renal cell carcinoma, chronic lymphocytic leukemia,hepatocellular carcinoma, non-small cell and small cell lung cancer,Non-Hodgkin lymphoma, acute myeloid leukemia, ovarian cancer, pancreaticcancer, prostate cancer, esophageal cancer including cancer of thegastric-esophageal junction, gallbladder cancer and cholangiocarcinoma,melanoma, gastric cancer, urinary bladder cancer, head and neck squamouscell carcinoma, or uterine cancer samples from patients and blood fromhealthy donors were analyzed in a stepwise approach:

1. HLA ligands from the malignant material were identified by massspectrometry

2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis wasused to identify genes over-expressed in the malignant tissue comparedwith a range of normal organs and tissues

3. Identified HLA ligands were compared to gene expression data.Peptides over-presented or selectively presented on tumor tissue,preferably encoded by selectively expressed or over-expressed genes asdetected in step 2 were considered suitable TUMAP candidates for amulti-peptide vaccine.

4. Literature research was performed in order to identify additionalevidence supporting the relevance of the identified peptides as TUMAPs

5. The relevance of over-expression at the mRNA level was confirmed byredetection of selected TUMAPs from step 3 on tumor tissue and lack of(or infrequent) detection on healthy tissues.

6. In order to assess, whether an induction of in vivo T-cell responsesby the selected peptides may be feasible, in vitro immunogenicity assayswere performed using human T cells from healthy donors as well as fromcancer patients.

In an aspect, the peptides are pre-screened for immunogenicity beforebeing included in the warehouse. By way of example, and not limitation,the immunogenicity of the peptides included in the warehouse isdetermined by a method comprising in vitro T-cell priming throughrepeated stimulations of CD8+ T cells from healthy donors withartificial antigen presenting cells loaded with peptide/MHC complexesand anti-CD28 antibody.

This method is preferred for rare cancers and patients with a rareexpression profile. In contrast to multi-peptide cocktails with a fixedcomposition as currently developed, the warehouse allows a significantlyhigher matching of the actual expression of antigens in the tumor withthe vaccine. Selected single or combinations of several “off-the-shelf”peptides will be used for each patient in a multitarget approach. Intheory an approach based on selection of e.g. 5 different antigenicpeptides from a library of 50 would already lead to approximately 17million possible drug product (DP) compositions.

In an aspect, the peptides are selected for inclusion in the vaccinebased on their suitability for the individual patient based on themethod according to the present invention as described herein, or asbelow.

The HLA phenotype, transcriptomic and peptidomic data is gathered fromthe patient's tumor material, and blood samples to identify the mostsuitable peptides for each patient containing “warehouse” andpatient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen,which are selectively or over-expressed in the patients tumor and, wherepossible, show strong in vitro immunogenicity if tested with thepatients' individual PBMCs.

Preferably, the peptides included in the vaccine are identified by amethod comprising: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; (b) comparingthe peptides identified in (a) with a warehouse (database) of peptidesas described above; and (c) selecting at least one peptide from thewarehouse (database) that correlates with a tumor-associated peptideidentified in the patient. For example, the TUMAPs presented by thetumor sample are identified by: (a1) comparing expression data from thetumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. Preferably, the sequences of MHCligands are identified by eluting bound peptides from MHC moleculesisolated from the tumor sample, and sequencing the eluted ligands.Preferably, the tumor sample and the normal tissue are obtained from thesame patient.

In addition to, or as an alternative to, selecting peptides using awarehousing (database) model, TUMAPs may be identified in the patient denovo, and then included in the vaccine. As one example, candidate TUMAPsmay be identified in the patient by (a1) comparing expression data fromthe tumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. As another example, proteins may beidentified containing mutations that are unique to the tumor samplerelative to normal corresponding tissue from the individual patient, andTUMAPs can be identified that specifically target the mutation. Forexample, the genome of the tumor and of corresponding normal tissue canbe sequenced by whole genome sequencing: For discovery of non-synonymousmutations in the protein-coding regions of genes, genomic DNA and RNAare extracted from tumor tissues and normal non-mutated genomic germlineDNA is extracted from peripheral blood mononuclear cells (PBMCs). Theapplied NGS approach is confined to the re-sequencing of protein codingregions (exome re-sequencing). For this purpose, exonic DNA from humansamples is captured using vendor-supplied target enrichment kits,followed by sequencing with e.g. a HiSeq2000 (IIIumina). Additionally,tumor mRNA is sequenced for direct quantification of gene expression andvalidation that mutated genes are expressed in the patients' tumors. Theresultant millions of sequence reads are processed through softwarealgorithms. The output list contains mutations and gene expression.Tumor-specific somatic mutations are determined by comparison with thePBMC-derived germline variations and prioritized. The de novo identifiedpeptides can then be tested for immunogenicity as described above forthe warehouse, and candidate TUMAPs possessing suitable immunogenicityare selected for inclusion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient by the method asdescribed above; (b) comparing the peptides identified in a) with awarehouse of peptides that have been prescreened for immunogenicity andoverpresentation in tumors as compared to corresponding normal tissue;(c) selecting at least one peptide from the warehouse that correlateswith a tumor-associated peptide identified in the patient; and (d)optionally, selecting at least one peptide identified de novo in (a)confirming its immunogenicity.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; and (b)selecting at least one peptide identified de novo in (a) and confirmingits immunogenicity.

Once the peptides for a personalized peptide based vaccine are selected,the vaccine is produced. The vaccine preferably is a liquid formulationconsisting of the individual peptides dissolved in between 20-40% DMSO,preferably about 30-35% DMSO, such as about 33% DMSO.

Each peptide to be included into a product is dissolved in DMSO. Theconcentration of the single peptide solutions has to be chosen dependingon the number of peptides to be included into the product. The singlepeptide-DMSO solutions are mixed in equal parts to achieve a solutioncontaining all peptides to be included in the product with aconcentration of ˜2.5 mg/ml per peptide. The mixed solution is thendiluted 1:3 with water for injection to achieve a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution is filtered througha 0.22 μm sterile filter. The final bulk solution is obtained.

Final bulk solution is filled into vials and stored at −20° C. untiluse. One vial contains 700 μL solution, containing 0.578 mg of eachpeptide. Of this, 500 μL (approx. 400 μg per peptide) will be appliedfor intradermal injection.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from glioblastoma, breast cancer, colorectal cancer,renal cell carcinoma, chronic lymphocytic leukemia, hepatocellularcarcinoma, non-small cell and small cell lung cancer, Non-Hodgkinlymphoma, acute myeloid leukemia, ovarian cancer, pancreatic cancer,prostate cancer, esophageal cancer including cancer of thegastric-esophageal junction, gallbladder cancer and cholangiocarcinoma,melanoma, gastric cancer, urinary bladder cancer, head and neck squamouscell carcinoma, or uterine cancer cells and since it was determined thatthese peptides are not or at lower levels present in normal tissues,these peptides can be used to diagnose the presence of a cancer.

The presence of claimed peptides on tissue biopsies in blood samples canassist a pathologist in diagnosis of cancer. Detection of certainpeptides by means of antibodies, mass spectrometry or other methodsknown in the art can tell the pathologist that the tissue sample ismalignant or inflamed or generally diseased, or can be used as abiomarker for glioblastoma, breast cancer, colorectal cancer, renal cellcarcinoma, chronic lymphocytic leukemia, hepatocellular carcinoma,non-small cell and small cell lung cancer, Non-Hodgkin lymphoma, acutemyeloid leukemia, ovarian cancer, pancreatic cancer, prostate cancer,esophageal cancer including cancer of the gastric-esophageal junction,gallbladder cancer and cholangiocarcinoma, melanoma, gastric cancer,urinary bladder cancer, head and neck squamous cell carcinoma, oruterine cancer. Presence of groups of peptides can enable classificationor sub-classification of diseased tissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmuno-surveillance. Thus, presence of peptides shows that thismechanism is not exploited by the analyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides such as T cell responses orantibody responses against the peptide or the peptide complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate response markers in immunotherapy approaches aiming toinduce lymphocyte responses by different means, e.g. vaccination ofprotein, nucleic acids, autologous materials, adoptive transfer oflymphocytes. In gene therapy settings, lymphocyte responses againstpeptides can be considered in the assessment of side effects. Monitoringof lymphocyte responses might also be a valuable tool for follow-upexaminations of transplantation therapies, e.g. for the detection ofgraft versus host and host versus graft diseases.

The present invention will now be described in the following exampleswhich describe preferred embodiments thereof, and with reference to theaccompanying figures, nevertheless, without being limited thereto. Forthe purposes of the present invention, all references as cited hereinare incorporated by reference in their entireties.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A to 1T show the over-presentation of various peptides in normaltissues (white bars) and different cancers (black bars). FIG. 1A—MET,Peptide: GLIAGVVSI (SEQ ID NO.: 2)—Tissues from left to right: 4 celllines (1 kidney, 2 pancreatic, 1 melanoma), 24 cancer tissues (1 braincancer, 1 gallbladder cancer, 7 kidney cancers, 1 rectum cancer, 1 livercancer, 7 lung cancers, 2 stomach cancers, 4 urinary bladder cancers);FIG. 1B)—TMEM223, Peptide: GLLFSLRSV (SEQ ID NO.: 92)—Tissues from leftto right: 2 cell lines (2 pancreatic), 1 normal tissue (1 lymph node),14 cancer tissues (4 leukocytic leukemia cancers, 2 myeloid cellscancers, 1 bone marrow cancer, 1 breast cancer, 1 lymph node cancer, 1ovarian cancer, 2 prostate cancers, 1 skin cancer, 1 urinary bladdercancer); FIG. 1C—PRKDC, Peptide: HYSQELSLLYL (SEQ ID NO.: 158)—Tissuesfrom left to right: 19 cancer tissues (1 brain cancer, 1 kidney cancer,2 liver cancers, 9 lung cancers, 2 prostate cancers, 4 stomach cancers);FIG. 1D—GPX6, GPX7, Peptide: TYSVSFPMF (SEQ ID NO.: 195)—Tissues fromleft to right: 3 cell lines (3 benign prostate hyperplasias), 1 normaltissue (1 stomach), 58 cancer tissues (1 brain cancer, 2 liver cancers,47 lung cancers, 7 prostate cancers, 1 stomach cancer). FIG. 1E to Rshow the over-presentation of various peptides in different cancertissues compared to normal tissues. The analyses included data from morethan 490 A*02 positive normal tissue samples and 70 A*24 positive normaltissue samples, 543 A*02 positive cancer samples and 200 A*24 positivecancer samples. Shown are only samples where the peptide was found to bepresented. FIG. 1E) Gene symbol: FEN1, Peptide: SIYQFLIAV (SEQ ID NO:8)—Tissues from left to right: 2 cell lines (1 blood cells, 1 pancreas),14 cancer tissues (3 leukocytic leukemia cancers, 1 myeloid cellscancer, 1 breast cancer, 1 gallbladder cancer, 1 head-and-neck cancer, 1colon cancer, 2 lung cancers, 3 lymph node cancers, 1 uterus cancer);FIG. 1F) Gene symbol: DERL3, Peptide: ALMAMLVYV (SEQ ID NO: 13)—Tissuesfrom left to right: 17 cancer tissues (1 bile duct cancer, 2 breastcancers, 1 gallbladder cancer, 3 head-and-neck cancers, 7 lung cancers,1 lymph node cancer, 1 ovarian cancer, 1 stomach cancer); FIG. 1G) Genesymbol: HEATR2, Peptide: ALAPHLDDA (SEQ ID NO: 237)—Tissues from left toright: 1 cell lines (blood cells), 10 cancer tissues (1 myeloid cellscancer, 2 brain cancers, 1 breast cancer, 1 lung cancer, 1 ovariancancer, 1 skin cancer, 1 urinary bladder cancer, 2 uterus cancers); FIG.1H) Gene symbol: SLC4A11, Peptide: ILLPRIIEA (SEQ ID NO: 259)—Tissuesfrom left to right: 1 cell lines (1 pancreas), 27 cancer tissues (3leukocytic leukemia cancers, 1 brain cancer, 2 breast cancers, 3head-and-neck cancers, 1 colon cancer, 1 rectum cancer, 6 lung cancers,5 ovarian cancers, 1 pancreas cancer, 2 skin cancers, 1 stomach cancer,1 uterus cancer); FIG. 1I) Gene symbol: ABCC11, Peptide: HLLEGSVGV (SEQID NO: 39)—Tissues from left to right: 7 cancer tissues (5 breastcancers, 1 liver cancer, 1 skin cancer); FIG. 1J) Gene symbol: PRAME,Peptide: VQLDSIEDLEV (SEQ ID NO: 32)—Tissues from left to right: 10cancer tissues (1 leukocytic leukemia cancer, 1 lung cancer, 4 ovariancancers, 3 skin cancers, 1 uterus cancer); FIG. 1K) Gene symbol: ZWILCH,Peptide: FYSRLLQKF (SEQ ID NO: 203)—Tissues from left to right: 1 cellline (1 benign prostate hyperplasia), 15 cancer tissues (13 lungcancers, 2 stomach cancers); FIG. 1L) Gene symbol: PRC1, Peptide:NYYEVHKELF (SEQ ID NO: 270)—Tissues from left to right: 14 cancertissues (1 brain cancer, 11 lung cancers, 2 stomach cancers); FIG. 1M)Gene symbol: GZMK, Peptide: KFSSFSLFF (SEQ ID NO: 164)—Tissues from leftto right: 2 cell lines (2 benign prostate hyperplasias), 14 cancertissues (11 lung cancers, 1 prostate cancer, 2 stomach cancers); FIG.1N) Gene symbols: TREX2, HAUS7, Peptide: LYITEPKTI (SEQ ID NO:287)—Tissues from left to right: 12 cancer tissues (3 brain cancers, 1liver cancer, 7 lung cancers, 1 stomach cancer); FIG. 1O) Gene symbol:DNMBP, Peptide: RYISDQLFTNF (SEQ ID NO: 278)—Tissues from left to right:1 normal tissue (1 lung), 31 cancer tissues (2 brain cancers, 1 kidneycancer, 2 liver cancers, 20 lung cancers, 2 prostate cancers, 4 stomachcancers); FIG. 1P) Gene symbol: PTK7, Peptide: VYQGHTALL (SEQ ID NO:277)—Tissues from left to right: 2 cell line (2 benign prostatehyperplasias), 4 normal tissues (1 rectum, 2 lungs, 1 pancreas), 64cancer tissues (5 brain cancers, 48 lung cancers, 6 prostate cancers, 5stomach cancers); FIG. 1Q) Gene symbols: NUP210P1, NUP210, Peptide:VYVSDIQEL (SEQ ID NO: 288)—Tissues from left to right: 2 normal tissues(1 colon, 1 pituitary gland), 21 cancer tissues (1 liver cancer, 14 lungcancers, 1 prostate cancer, 5 stomach cancers); FIG. 1R) Gene symbol:ATAD2, Peptide: VYTLDIPVL (SEQ ID NO: 160)—Tissues from left to right:17 cancer tissues (1 liver cancer, 9 lung cancers, 1 prostate cancer, 6stomach cancers); FIG. 1S) Gene symbol: DNTT, Peptide: KLFTSVFGV (SEQ IDNO: 305)—Tissues from left to right: 5 cancer tissues (5 blood cellscancers); FIG. 1T) Gene symbol: AR, Peptide: ALLSSLNEL (SEQ ID NO:306)—Tissues from left to right: 3 cell lines (1 kidney, 2 prostates), 4normal tissues (1 liver, 1 lung, 1 ovary, 1 uterus), 29 cancer tissues(1 bile duct cancer, 2 blood cells cancers, 1 brain cancer, 3 breastcancers, 1 kidney cancer, 5 liver cancers, 1 lung cancer, 1 lymph nodecancer, 4 ovary cancers, 6 prostate cancers, 1 urinary bladder cancer, 3uterus cancers).

FIGS. 2A to 2I show exemplary expression profiles of source genes of thepresent invention that are highly over-expressed or exclusivelyexpressed in different cancers in a panel of normal tissues (white bars)and different cancers samples (black bars). FIG. 2A—TNC, Peptide:KLLDPQEFTL, (SEQ ID NO.: 59)—Tissues from left to right: 73 normaltissue samples (6 arteries, 1 blood cells, 1 brain, 1 heart, 2 livers, 2lungs, 2 veins, 1 adipose tissue, 1 adrenal gland, 6 bone marrows, 1cartilage, 1 colon, 1 esophagus, 2 eyes, 2 gallbladders, 1 kidney, 6lymph nodes, 5 pancreases, 2 pituitary glands, 1 rectum, 1 salivarygland, 1 skeletal muscle, 1 skin, 1 small intestine, 1 spleen, 1stomach, 1 thyroid gland, 7 tracheas, 1 urinary bladder, 1 breast, 5ovaries, 3 placentas, 1 prostate, 1 testis, 1 thymus, 1 uterus) and 46cancer samples (24 brain cancers, 11 lung cancers, 11 esophaguscancers); FIG. 2B—LRRC15, Peptide: ILNTHITEL, (SEQ ID NO.: 149)—Tissuesfrom left to right: 73 normal tissue samples (6 arteries, 1 blood cells,1 brain, 1 heart, 2 livers, 2 lungs, 2 veins, 1 adipose tissue, 1adrenal gland, 6 bone marrows, 1 cartilage, 1 colon, 1 esophagus, 2eyes, 2 gallbladders, 1 kidney, 6 lymph nodes, 5 pancreases, 2 pituitaryglands, 1 rectum, 1 salivary gland, 1 skeletal muscle, 1 skin, 1 smallintestine, 1 spleen, 1 stomach, 1 thyroid gland, 7 tracheas, 1 urinarybladder, 1 breast, 5 ovaries, 3 placentas, 1 prostate, 1 testis, 1thymus, 1 uterus) and 56 cancer samples (10 breast cancers, 3gallbladder cancers, 11 stomach cancers, 10 lymph node cancers, 11 lungcancers, 11 esophagus cancers); FIG. 2C—C1QL1, Peptide: TYTTVPRVAF, (SEQID NO.: 172)—Tissues from left to right: 73 normal tissue samples (6arteries, 1 blood cells, 1 brain, 1 heart, 2 livers, 2 lungs, 2 veins, 1adipose tissue, 1 adrenal gland, 6 bone marrows, 1 cartilage, 1 colon, 1esophagus, 2 eyes, 2 gallbladders, 1 kidney, 6 lymph nodes, 5pancreases, 2 pituitary glands, 1 rectum, 1 salivary gland, 1 skeletalmuscle, 1 skin, 1 small intestine, 1 spleen, 1 stomach, 1 thyroid gland,7 tracheas, 1 urinary bladder, 1 breast, 5 ovaries, 3 placentas, 1prostate, 1 testis, 1 thymus, 1 uterus) and 34 cancer samples (24 braincancers, 10 kidney cancers); FIG. 2D—AMC2, Peptide: GYIDNVTLI, (SEQ IDNO.: 220)—Tissues from left to right: 73 normal tissue samples (6arteries, 1 blood cells, 1 brain, 1 heart, 2 livers, 2 lungs, 2 veins, 1adipose tissue, 1 adrenal gland, 6 bone marrows, 1 cartilage, 1 colon, 1esophagus, 2 eyes, 2 gallbladders, 1 kidney, 6 lymph nodes, 5pancreases, 2 pituitary glands, 1 rectum, 1 salivary gland, 1 skeletalmuscle, 1 skin, 1 small intestine, 1 spleen, 1 stomach, 1 thyroid gland,7 tracheas, 1 urinary bladder, 1 breast, 5 ovaries, 3 placentas, 1prostate, 1 testis, 1 thymus, 1 uterus) and 48 cancer samples (11 lungcancers, 11 esophagus cancers, 26 pancreas cancers). FIG. 2E—ABCC11,Peptide: HLLEGSVGV, (SEQ ID NO.: 39)—Tissues from left to right: 6arteries, 2 blood cellss, 6 brains, 4 hearts, 8 livers, 6 lungs, 2veins, 1 adipose tissue, 5 adrenal glands, 1 bile duct, 5 bone marrows,1 cartilage, 1 chest wall and skeletal muscle, 1 colon, 5 esophagi, 2eyes, 2 gallbladders, 8 head-and-necks, 5 head-and-neck and salivaryglands, 1 kidney, 6 lymph nodes, 4 pancreases, 3 parathyroid glands, 2peripheral nerves, 3 peritoneums, 2 pituitary glands, 3 pleuras, 1rectum, 1 skeletal muscle, 2 skins, 1 small intestine, 1 spleen, 1stomach, 1 thyroid gland, 7 tracheas, 5 ureters, 1 urinary bladder, 1breast, 5 ovaries, 5 placentas, 1 prostate, 1 testis, 2 thymi, 1 uterus,and 10 breast cancers samples. FIG. 2F—PRAME, Peptide: VQLDSIEDLEV, (SEQID NO.: 32)—Tissues from left to right: 6 arteries, 2 blood cellss, 6brains, 4 hearts, 8 livers, 6 lungs, 2 veins, 1 adipose tissue, 5adrenal glands, 1 bile duct, 5 bone marrows, 1 cartilage, 1 chest walland skeletal muscle, 1 colon, 5 esophagi, 2 eyes, 2 gallbladders, 8head-and-necks, 5 head-and-neck and salivary glands, 1 kidney, 6 lymphnodes, 4 pancreases, 3 parathyroid glands, 2 peripheral nerves, 3peritoneums, 2 pituitary glands, 3 pleuras, 1 rectum, 1 skeletal muscle,2 skins, 1 small intestine, 1 spleen, 1 stomach, 1 thyroid gland, 7tracheas, 5 ureters, 1 urinary bladder, 1 breast, 5 ovaries, 5placentas, 1 prostate, 1 testis, 2 thymi, 1 uterus, and 37 cancersamples (10 melanoma cancers, 17 ovarian cancers, 10 uterine cancers).FIG. 2G—SPINK2, Peptide: ALSVLRLAL, (SEQ ID NO.: 251)—Tissues from leftto right: 6 arteries, 2 blood cellss, 6 brains, 4 hearts, 8 livers, 6lungs, 2 veins, 1 adipose tissue, 5 adrenal glands, 1 bile duct, 5 bonemarrows, 1 cartilage, 1 chest wall and skeletal muscle, 1 colon, 5esophagi, 2 eyes, 2 gallbladders, 8 head-and-necks, 5 head-and-neck andsalivary glands, 1 kidney, 6 lymph nodes, 4 pancreases, 3 parathyroidglands, 2 peripheral nerves, 3 peritoneums, 2 pituitary glands, 3pleuras, 1 rectum, 1 skeletal muscle, 2 skins, 1 small intestine, 1spleen, 1 stomach, 1 thyroid gland, 7 tracheas, 5 ureters, 1 urinarybladder, 1 breast, 5 ovaries, 5 placentas, 1 prostate, 1 testis, 2thymi, 1 uterus, and 11 acute myeloid leukemia samples. FIG. 2H—MAGEC2,Peptide: TLDEKVAEL, (SEQ ID NO.: 24)—Tissues from left to right: 6arteries, 2 blood cellss, 6 brains, 4 hearts, 8 livers, 6 lungs, 2veins, 1 adipose tissue, 5 adrenal glands, 1 bile duct, 5 bone marrows,1 cartilage, 1 chest wall and skeletal muscle, 1 colon, 5 esophagi, 2eyes, 2 gallbladders, 8 head-and-necks, 5 head-and-neck and salivaryglands, 1 kidney, 6 lymph nodes, 4 pancreases, 3 parathyroid glands, 2peripheral nerves, 3 peritoneums, 2 pituitary glands, 3 pleuras, 1rectum, 1 skeletal muscle, 2 skins, 1 small intestine, 1 spleen, 1stomach, 1 thyroid gland, 7 tracheas, 5 ureters, 1 urinary bladder, 1breast, 5 ovaries, 5 placentas, 1 prostate, 1 testis, 2 thymi, 1 uterus,and 29 cancer samples (19 liver cancers, 10 melanoma cancers). FIG.2I—C1orf186, Peptide: FLTAINYLL, (SEQ ID NO.: 72)—Tissues from left toright: 6 arteries, 2 blood cellss, 6 brains, 4 hearts, 8 livers, 6lungs, 2 veins, 1 adipose tissue, 5 adrenal glands, 1 bile duct, 5 bonemarrows, 1 cartilage, 1 chest wall and skeletal muscle, 1 colon, 5esophagi, 2 eyes, 2 gallbladders, 8 head-and-necks, 5 head-and-neck andsalivary glands, 1 kidney, 6 lymph nodes, 4 pancreases, 3 parathyroidglands, 2 peripheral nerves, 3 peritoneums, 2 pituitary glands, 3pleuras, 1 rectum, 1 skeletal muscle, 2 skins, 1 small intestine, 1spleen, 1 stomach, 1 thyroid gland, 7 tracheas, 5 ureters, 1 urinarybladder, 1 breast, 5 ovaries, 5 placentas, 1 prostate, 1 testis, 2thymi, 1 uterus, and 49 cancer samples (11 acute myeloid leukemiasamples, 17 ovarian cancer samples, 11 renal cell carcinoma samples, 10uterine cancer samples).

FIGS. 3A and 3B show exemplary immunogenicity data: flow cytometryresults after peptide-specific multimer staining.

FIGS. 4A to 4D show exemplary results of peptide-specific in vitro CD8+T cell responses of a healthy HLA-A*02+ donor. CD8+ T cells were primedusing artificial APCs coated with anti-CD28 mAb and HLA-A*02 in complexwith SeqID No 27 peptide (FIG. 4A, left panel), SeqID No 229 peptide(FIG. 4B, left panel), SeqID No 230 peptide (FIG. 4C, left panel) andSeqID No 235 peptide (FIG. 4D, left panel), respectively. After threecycles of stimulation, the detection of peptide-reactive cells wasperformed by 2D multimer staining with A*02/SeqID No 27 (FIG. 4A),A*02/SeqID No 229 (FIG. 4B), A*02/SeqID No 230 (FIG. 4C) or A*02/SeqIDNo 235 (FIG. 4D). Right panels (FIGS. 4A, 4B, 4C and 4D) show controlstaining of cells stimulated with irrelevant A*02/peptide complexes.Viable singlet cells were gated for CD8+ lymphocytes. Boolean gateshelped excluding false-positive events detected with multimers specificfor different peptides. Frequencies of specific multimer+ cells amongCD8+ lymphocytes are indicated.

FIGS. 5A to 5C show exemplary results of peptide-specific in vitro CD8+T cell responses of a healthy HLA-A*24+ donor. CD8+ T cells were primedusing artificial APCs coated with anti-CD28 mAb and HLA-A*24 in complexwith SeqID No 159 peptide (FIG. 5A, left panel), SeqID No 161 peptide(FIG. 5B, left panel) and SeqID No 281 peptide (FIG. 5C, left panel),respectively. After three cycles of stimulation, the detection ofpeptide-reactive cells was performed by 2D multimer staining withA*24/SeqID No 159 (FIG. 5A), A*24/SeqID No 161 (FIG. 5B) or A*24/SeqIDNo 281 (FIG. 5C). Right panels (FIGS. 5A, 5B and 5C) show controlstaining of cells stimulated with irrelevant A*24/peptide complexes.Viable singlet cells were gated for CD8+ lymphocytes. Boolean gateshelped excluding false-positive events detected with multimers specificfor different peptides. Frequencies of specific multimer+ cells amongCD8+ lymphocytes are indicated.

EXAMPLES Example 1

Identification and Quantitation of Tumor Associated Peptides Presentedon the Cell Surface

Tissue Samples

Patients' tumor tissues were obtained from:

Asterand (Detroit, Mich., USA & Royston, Herts, UK); Bio-Options Inc.(Brea, Calif., USA); BioServe (Beltsville, Md., USA); Center for cancerimmune therapy (CCIT), Herlev Hospital (Herlev, Denmark); GeneticistInc. (Glendale, Calif., USA); Indivumed GmbH (Hamburg, Germany);Istituto Nazionale Tumori “Pascale” (Naples, Italy); Kyoto PrefecturalUniversity of Medicine (KPUM) (Kyoto, Japan); Leiden University MedicalCenter (LUMC) (Leiden, Netherlands); ProteoGenex Inc. (Culver City,Calif., USA); Saint Savas Hospital (Athens, Greece); Stanford CancerCenter (Stanford, Calif., USA); Tissue Solutions Ltd (Glasgow, UK);University Hospital Bonn (Bonn, Germany); University Hospital Geneva(Geneva, Switzerland); University Hospital Heidelberg (Heidelberg,Germany); University Hospital Munich (Munich, Germany); Osaka CityUniversity (OCU) (Osaka, Japan); University Hospital Tübingen (Tübingen,Germany); Val d'Hebron University Hospital (Barcelona, Spain).

Normal Tissues were Obtained from

Asterand (Detroit, Mich., USA & Royston, Herts, UK); BioCat GmbH(Heidelberg, Germany); Bio-Options Inc. (Brea, Calif., USA); BioServe(Beltsville, Md., USA); Capital BioScience Inc. (Rockville, Md., USA);Geneticist Inc. (Glendale, Calif., USA); Kyoto Prefectural University ofMedicine (KPUM) (Kyoto, Japan); ProteoGenex Inc. (Culver City, Calif.,USA); Tissue Solutions Ltd (Glasgow, UK); University Hospital Geneva(Geneva, Switzerland); University Hospital Heidelberg (Heidelberg,Germany); University Hospital Munich (Munich, Germany); Osaka CityUniversity (OCU) (Osaka, Japan); University Hospital Tübingen (Tübingen,Germany).

Written informed consents of all patients had been given before surgeryor autopsy. Tissues were shock-frozen immediately after excision andstored until isolation of TUMAPs at −70° C. or below.

Peptides were selected if three conditions were true: (1) Its underlyingtranscript(s) and/or exon(s) are expressed at low levels, i.e. themedian reads per kilobase per million reads (RPKM) was required to beless than 10 for the following organs: brain, blood vessel, heart,liver, lung, blood. In addition, the median RPKM was required to be lessthan 50 for the following organs: urinary bladder, salivary gland,stomach, adrenal gland, colon, small intestine, spleen, bone marrow,pancreas, muscle, adipose tissue, skin, esophagus, kidney, thyroidgland, pituitary gland, nerve. (2) Its underlying transcript(s) and/orexon(s) are considered over-expressed if the highest 90% percentile ofexpression level in a tumor sample (based on data generated by the TCGAResearch Network: www.cancergenome.nih.gov/) was more than 6-fold abovethe highest 75% percentile of expression levels determined from normalsamples based on a database of RNASeq data covering around 3000 normaltissue samples (Lonsdale, 2013). (3) The peptide was tumor-associated,i.e. found specifically or on tumors or over-expressed compared to abaseline of normal tissues (cf. Example 1).

Sample numbers for HLA-A*02 TUMAP selection were: for pancreatic cancerN=16, for renal cancer N=20, for colorectal cancer N=28, for esophagealcarcinoma including cancer of the gastric-esophageal junction N=17, forprostate tumors N=39, for hepatocellular carcinoma N=16, for non-smallcell lung cancer N=90, for gastric cancer N=29, for breast cancer N=17,for melanoma N=7, for ovarian cancer N=20, for chronic lymphocyticleukemia N=17, for urinary bladder cancer N=16, for small-cell lungcancer N=19, for gallbladder cancer and cholangiocarcinoma N=6, foracute myeloid leukemia N=18, for glioblastoma N=41, for testis cancerN=1, for Non-Hodgkin lymphoma N=18, for uterine cancer N=15, and fornormal tissues N=262.

Sample numbers for HLA-A*24 TUMAP selection were: for gastric cancerN=44, for prostate tumors N=40, for non-small cell lung cancer N=91, forhepatocellular carcinoma N=15, for renal cancer N=2, for colorectalcancer N=1, for glioblastoma N=18 and for normal tissues N=70.

FIG. 1 also show results for cell lines including kidney cancer celllines, melanoma cell lines, pancreatic cancer cell lines and benignprostate hyperplasias.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to a slightly modifiedprotocol (Falk et al., 1991; Seeger et al., 1999) using theHLA-A*02-specific antibody BB7.2, the HLA-A, -B, C-specific antibodyW6/32, CNBr-activated sepharose, acid treatment, and ultrafiltration.

Mass Spectrometry Analyses

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (nanoAcquity UPLCsystem, Waters) and the eluting peptides were analyzed in LTQ-velos andfusion hybrid mass spectrometers (ThermoElectron) equipped with an ESIsource. Peptide pools were loaded directly onto the analyticalfused-silica micro-capillary column (75 μm i.d.×250 mm) packed with 1.7μm C18 reversed-phase material (Waters) applying a flow rate of 400 nLper minute. Subsequently, the peptides were separated using a two-step180 minute-binary gradient from 10% to 33% B at a flow rate of 300 nLper minute. The gradient was composed of Solvent A (0.1% formic acid inwater) and solvent B (0.1% formic acid in acetonitrile). A gold coatedglass capillary (PicoTip, New Objective) was used for introduction intothe nanoESl source. The LTQ-Orbitrap mass spectrometers were operated inthe data-dependent mode using a TOPS strategy. In brief, a scan cyclewas initiated with a full scan of high mass accuracy in the Orbitrap(R=30 000), which was followed by MS/MS scans also in the Orbitrap(R=7500) on the five most abundant precursor ions with dynamic exclusionof previously selected ions. Tandem mass spectra were interpreted bySEQUEST and additional manual control. The identified peptide sequencewas assured by comparison of the generated natural peptide fragmentationpattern with the fragmentation pattern of a synthetic sequence-identicalreference peptide.

Label-free relative LC-MS quantitation was performed by ion countingi.e. by extraction and analysis of LC-MS features (Mueller et al.,2007). The method assumes that the peptide's LC-MS signal areacorrelates with its abundance in the sample. Extracted features werefurther processed by charge state deconvolution and retention timealignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MSfeatures were cross-referenced with the sequence identification resultsto combine quantitative data of different samples and tissues to peptidepresentation profiles. The quantitative data were normalized in atwo-tier fashion according to central tendency to account for variationwithin technical and biological replicates. Thus each identified peptidecan be associated with quantitative data allowing relativequantification between samples and tissues. In addition, allquantitative data acquired for peptide candidates was inspected manuallyto assure data consistency and to verify the accuracy of the automatedanalysis. For each peptide a presentation profile was calculated showingthe mean sample presentation as well as replicate variations. Theprofiles juxtapose different cancer samples to a baseline of normaltissue samples.

Presentation profiles of exemplary over-presented peptides are shown inFIG. 1. Presentation scores for exemplary peptides are shown in Table 15and Table 16.

Table 13 (A and B) and Table 14 (A and B) show the presentation onvarious cancer entities for selected peptides, and thus the particularrelevance of the peptides as mentioned for the diagnosis and/ortreatment of the cancers as indicated (e.g. peptide SEQ ID No. 1 forglioblastoma, non-small cell lung cancer, and ovarian cancer, peptideSEQ ID No. 2 for glioblastoma, gastric cancer, non-small cell lungcancer, urinary bladder cancer, gallbladder adenocarcinoma andcholangiocarcinoma, renal cell carcinoma, colorectal cancer andpancreatic cancer).

TABLE 13A Overview of presentation of selectedHLA-A*02-binding tumor-associated peptides of the present inventionacross entities. SEQ Peptide Presentation ID No. Sequenceon cancer entities   1 LLYPEPWSV GB, NSCLC, OC   2 GLIAGVVSIGB, GC, NSCLC, UBC, GBC_CCC, RCC, CRC, PC   3 KLEENGDLYLNSCLC, OC, SCLC, UBC, BRCA, GBC_CCC, MEL, NHL   4 KLMPGTYTLNSCLC, OC, UEC, AML, NHL   5 GIVAHIQEV GC, NSCLC, PCA, CLL, OSCAR,OC, CRC, AML, NHL   6 ALFDSLRHV GB   7 ILDHEVPSLNSCLC, CLL, OSCAR, OC, UBC, MEL, RCC, AML,   8 SIYQFLIAVNSCLC, CLL, GBC_CCC, AML, NHL   9 FLVDGSYSI NSCLC, OSCAR, GBC_CCC, CRC 10 GIAGSLKTV NSCLC, OSCAR, UBC, UEC, CRC, AML  11 ALSPSYLTVGC, NSCLC, CLL, BRCA, GBC_CCC, NHL  12 GLLPLLHRA GB, NSCLC, UEC, RCC  13ALMAMLVYV GC, NSCLC, OC, SCLC, BRCA, GBC_CCC  14 ILAKDLFEINSCLC, HCC, OSCAR, UBC, AML, NHL  15 YLDLSHNQL NSCLC, CLL, BRCA, GBC_CCC 16 YTLDIPVLFGV NSCLC, HCC, CLL, MEL, NHL  17 AVFPDDMPTLGC, NSCLC, OSCAR, OC, NHL  18 ILLDLTDNRL NSCLC, OSCAR, UBC, BRCA, MEL,RCC, CRC  19 SISDNVWEV GB, OSCAR, OC, UEC, BRCA, MEL, NHL  20 GLSQITNQLCLL, UBC, NHL  21 AIQDEIRSV GB, NSCLC, OSCAR, OC, BRCA, NHL  22FVDPNTQEKV GC, HCC, OSCAR, OC, UBC, BRCA,  23 SLFSDEFKVNSCLC, OSCAR, UBC, RCC, CRC, AML,  24 TLDEKVAEL NSCLC, HCC, OSCAR, MEL, 25 TMDSVLVTV OC, UEC  26 ALQEELTEL BRCA, GBC_CCC, MEL, AML, NHL  27RLMEENWNA NSCLC, OSCAR, OC, UEC, CRC  28 SLPNGKPVSV NSCLC, OC, UBC, NHL 29 YLLDPSITL NSCLC, CLL, BRCA, GBC_CCC, AML, NHL  30 AMIEEVFEAPCA, OC, SCLC, UBC, BRCA, MEL  31 TITETTVEV GB  32 VQLDSIEDLEV CLL, OC 33 YIKTELISV GC, NSCLC, OSCAR, OC, NHL  34 FLLATEVVTVOC, SCLC, AML, NHL  35 FLLPFSTVYL NSCLC, CLL, OC, AML, NHL  36SLADTNSLAVV SCLC, MEL  37 ILAPFSVDL AML  38 FLGPRIIGL CLL, AML, NHL  39HLLEGSVGV BRCA  40 VLIDPQWVLTA OC, NHL  41 ALFENTPKA GB  42 LLDSVSRLNSCLC, GBC_CCC, CRC  43 KAIEVLLTL GC, OSCAR, AML  44 SLFETAWEANSCLC, MEL, AML, NHL  45 SLTEVSLPL OC, GBC_CCC, AML, NHL  46 SQFPLPLAVGC, NSCLC, OSCAR, UBC  47 ALLERGELFV NSCLC, CLL, OC, NHL  48 QVIEDSTGVGC, NSCLC, OSCAR, OC  49 ALNIATHVL NSCLC, CLL, UBC, NHL  50 ILFHGVFYANSCLC, OC, UBC, BRCA  51 LLFSRLCGA GB, NSCLC  52 RLAVLFSGAGC, NSCLC, OSCAR, OC  53 KMVGLVVAI CLL, AML, NHL  54 VLNPLITAVNSCLC, PCA, AML, NHL  55 SLATKIVEA PCA, OC, BRCA, AML  56 FLHDEKEGIYINSCLC, OC, NHL  57 TVFTDHMLTV NSCLC, OSCAR, OC, NHL  58 YLLPLLPAL GB  59KLLDPQEFTL GB, NSCLC  60 ALFAPLVHL AML  61 AIVKEIVNI GC, NSCLC, OSCAR 62 ALNPELVQA GB, NSCLC, RCC  63 SQIPAQPSV GC, NSCLC, OSCAR  64SLFPDSLIV PCA  65 SVVPDVRSV GC, NSCLC, OSCAR  66 KLIFSVEAVNSCLC, SCLC, UEC, BRCA  67 TLLQRLTEV NSCLC, CLL, RCC, AML  68 SLSNRLYYLGC, OSCAR, CRC  69 FLAVGLVDV AML, NHL  70 LLLGDSALYL RCC, NHL  71VLHSKFWVV CLL, NHL  72 FLTAINYLL OC, RCC  73 YTLREVDTV NSCLC, OSCAR, OC 74 TLFGYSVVL AML  75 AVIKFLELL GC, NSCLC, OSCAR  76 AVGPVHNSV GC, OSCAR 77 TLIDEQDIPLV NSCLC, OC, SCLC  78 TVVTRLDEI GC, OSCAR, OC  79VTFKEYVTV GC, NSCLC, OSCAR  80 KLYEADFVL NSCLC, AML, NHL  81 NALDKVLSVGC, NSCLC, OSCAR  82 FIFDEAEKL GC, NSCLC, OSCAR  83 GQASYFYVA CRC, AML 84 ALCPRIHEV GB  85 VLNDILVRA OC, UEC  86 SVDSHFQEV GC, OSCAR, OC  87TIYKDFVYI GC, NSCLC, OSCAR  88 AQADHLPQL GC, NSCLC, OSCAR  89 QLAPVFQRVCLL, OC, NHL  90 FLQDLEQRL GC, NSCLC, PCA, CLL, OSCAR,OC, SCLC, UBC, UEC, BRCA, GBC_CCC, CRC, NHL  91 KLFDESILIGC, NSCLC, PCA, CLL, OSCAR, OC, SCLC, UEC, BRCA, AML, NHL  92 GLLFSLRSVNSCLC, HCC, PCA, CLL, OSCAR, OC, UBC, BRCA, MEL, RCC, CRC,  AML, NHL  93QVLELDVADI GC, NSCLC, PCA, SCLC, UBC, UEC, BRCA, CRC, AML, NHL  94LLLPAVPVGA GB, NSCLC, OC, UBC, CRC, AML  95 GLLGSLFFLNSCLC, OC, SCLC, UEC, BRCA, GBC_CCC, AML, NHL  96 LLVSHLYLVNSCLC, CLL, OC, UBC, NHL  97 STLPKSLSL GB, GC, NSCLC, HCC, PCA,OSCAR, OC, BRCA, NHL  98 RLFPDFFTRVAL NSCLC  99 YLLQSVNQLLLNSCLC, CLL, NHL 100 ALLGMIIVGV PCA, SCLC, BRCA 101 ALADFMLSL AML 102VLLDIQEVFQI AML 103 YLVSEIFKA AML 104 ALISWQPPRA GB 105 ALLGTKILL NHL106 FINDSIVYL AML 107 LLVPTSGIYFV NHL 108 ILLKNLVTI BRCA 109 SLDPSVTHLUEC 110 FLLGVSKEV MEL 111 AIVDLIHDI GC, NSCLC, OSCAR 112 SLGKFTFDVNSCLC, UEC 113 FLERGLESA GC, OSCAR, UBC 114 QLIQTLHAV OC, RCC 115SLDPDTLPAV NSCLC, OC 116 TIDESGSIL UBC, NHL 117 KMPDVELFV NSCLC 118QLWQFLVTL OC 119 FIIQGLRSVGA NSCLC, OC 120 VTPVTVSAV GC, OSCAR 121FTIFRTISV CLL 122 GVVDPVHGV GC, NSCLC, OSCAR 123 VLDPALPALV OC, UBC 124KVMATIEKV NSCLC, OC 125 SLADYEHFV GB, PCA 126 QMFQYFITV NSCLC, CLL 127KLDGNELDL NSCLC 128 TQSPATLSV GC, OSCAR 129 RLQDILWFL NSCLC, AML 130SLLGGTFVGI UBC 131 VTSNSGILGV CLL, NHL 132 ILGEVLAQL CLL, NHL 133ALLPRLHQL PCA 134 GLAVPTPSV NSCLC, OC 135 HLSTIIHEA NSCLC, CLL 136FLFGGVLMTL OC, NHL 137 EIASITEQL GB, NSCLC 138 ALLAKILQIGB, GC, NSCLC, HCC, PCA, CLL, OSCAR, OC, SCLC, UBC, BRCA,GBC_CCC, MEL, RCC, CRC, AML, NHL 139 FLLPTGAEA GC, NSCLC, HCC, PCA, CLL,OSCAR, OC, SCLC, UBC, UEC, BRCA, MEL, RCC, CRC, PC, AML, NHL 140VLLEELEAL NSCLC, PCA, CLL, OSCAR, OC, SCLC, UEC, BRCA, GBC_CCC,MEL, AML, NHL 141 FLDKVLVAA GC, NSCLC, PCA, CLL, OSCAR,UBC, RCC, AML, NHL 142 ILVEGISTV GB, NSCLC, PCA, OC, SCLC, BRCA, GBC_CCC143 ALLPELREV GB, GC, NSCLC, HCC, BRCA,  GBC_CCC, RCC, CRC 144 ALLAFFPGLGB, GC, NSCLC, OSCAR, OC, UBC, BRCA, MEL, CRC, PC, AML, NHL 145YLWATIQRI NSCLC, OSCAR, OC, AML 146 ALHFSEDEI PCA, UEC 147 YLMDDTVEIOC, GBC_CCC 148 MLAGIAITV GB, GBC_CCC 149 ILNTHITEL NHL 150 VLYDRPLKINSCLC, CLL, OSCAR, OC, MEL, RCC, CRC, NHL 151 SVLDSTAKVGC, NSCLC, OSCAR, OC, UBC, BRCA, RCC, CRC, NHL 152 MMVGDLLEVGB, GC, NSCLC, CLL, MEL, CRC, AML, NHL 153 FISERVEVVNSCLC, PCA, CLL, OSCAR, OC, UBC, NHL 154 RLLGTEFQV NSCLC, HCC, MEL, 155LLNPVVEFV CLL, OC, GBC_CCC, NHL 156 ILGDLSHLL NSCLC, PC 157 TLTSLLAQAUBC 229 AILAHLNTV GB, NSCLC, HCC, CLL, OSCAR, OC, SCLC, UBC, BRCA, CRC,AML, NHL 230 KLQNIMMLL NSCLC, HCC, CLL, UBC, BRCA,GBC_CCC, RCC, CRC, AML, NHL 231 MLDKYSHYL NSCLC, CLL, OSCAR, OC, NHL 232KIFPAALQLV NSCLC, HCC, CLL, OC, SCLC, UBC, UEC, GBC_CCC, CRC, NHL 233HLFDAFVSV NSCLC, UBC, BRCA, CRC, AML, NHL 234 LLSPHNPALNSCLC, CLL, OSCAR, OC, UBC, BRCA, NHL 235 KIIDFLSAL OC, UEC, AML, NHL236 STIAILNSV GB, GC, NSCLC, PCA, CLL, OSCAR, NHL 237 ALAPHLDDAGB, NSCLC, HCC, OC, UBC, UEC, BRCA, MEL 238 GLYERPTAANSCLC, PCA, PC, AML 239 KMNESTRSV NSCLC, GBC_CCC, NHL 240 YMGEEKLIASVNSCLC, CLL, OC, UBC, MEL, 241 KTIQQLETV NSCLC, OC, NHL 242 WLYGEDHQINSCLC, UEC, AML 243 FMADDIFSV NHL 244 YLLEKNRVV NSCLC, OSCAR, OC 245SLLDLPLSL CLL, NHL 246 TVSDVLNSV GB, NSCLC, CLL 247 ALYEGYATVNSCLC, HCC, CLL, OSCAR, OC, SCLC, UBC, UEC, BRCA,GBC_CCC, MEL, RCC, CRC, PC, NHL 248 YLDRFLAGV GB, NSCLC, PCA, CLL, CRC,AML, NHL 249 GLCERLVSL GB, NSCLC, CLL, RCC, CRC 250 SLAPATPEV MEL, NHL251 ALSVLRLAL AML 252 RLMEICESL AML 253 ALAELIDNSL CLL 254 KLQGKLPELNSCLC, NHL 255 SLLHFTENL AML, NHL 256 SLGEEQFSV MEL, NHL 257 GLYTDPCGVNSCLC, NHL 258 LLSERFINV HCC, PCA 259 ILLPRIIEA NSCLC, OC 260 ILLEKILSLNSCLC, CRC 261 QLQDRVYAL NSCLC, HCC, CLL, OSCAR, OC,SCLC, UBC, BRCA, RCC, CRC, NHL 262 FMVDKAIYL GC, NSCLC, PCA, OSCAR, OC,SCLC, BRCA, GBC_CCC, CRC, PC, NHL 263 VLLSEQGDVKLNSCLC, PCA, CLL, OSCAR, OC, MEL, NHL 264 KLFPQETLFLNSCLC, CLL, OSCAR, AML, NHL 265 NTCPYVHNI GB, CLL 266 YAIGLVMRLCLL, BRCA, AML 290 KIVDFSYSV GB, GC, NSCLC, HCC, OSCAR,OC, SCLC, UBC, UEC, BRCA, MEL, CRC, AML, NHL 291 KLDETGNSLGB, GC, NSCLC, HCC, CLL, OSCAR, OC, SCLC, UBC, UEC,MEL, RCC, CRC, PC, AML, NHL 292 GMMTAILGV GB, GC, NSCLC, HCC, PCA, CLL,UEC, GBC_CCC, RCC, CRC, PC, AML, NHL 293 FLVDGSWSI GB 294 GLMKYIGEV PCAGB = glioblastoma, BRCA = breast cancer, CRC = colorectal cancer, RCC =renal cell carcinoma, CLL = chronic lymphocytic leukemia, HCC =hepatocellular carcinoma, NSCLC = non-small cell lung cancer, SCLC =small cell lung cancer, NHL = non-Hodgkin lymphoma, AML = acute myeloidleukemia, OC = ovarian cancer, PC = pancreatic cancer, PCA = prostatecancer and benign prostate hyperplasia, OSCAR = esophageal cancer,including cancer of the gastric-esophageal junction, GBC_CCC =gallbladder adenocarcinoma and cholangiocarcinoma, MEL = melanoma, GC =gastric cancer, UBC = urinary bladder cancer, UEC = uterine cancer.

Tables 13B show the presentation on additional cancer entities forselected peptides, and thus the particular relevance of the peptides asmentioned for the diagnosis and/or treatment of the cancers asindicated.

TABLE 13BOverview of presentation of selected HLA-A*02-binding tumor-associatedpeptides of the present invention across entities. GB =glioblastoma, BRCA = breast cancer, CRC = colorectal cancer, RCC =renal cell carcinoma, CLL = chronic lymphocytic leukemia, HCC =hepatocellular carcinoma, NSCLC = non-small cell lung cancer, SCLC =small cell lung cancer, NHL = non-Hodgkin lymphoma, AML = acutemyeloid leukemia, OC = ovarian cancer, PC = pancreatic cancer, PCA =prostate cancer and benign prostate hyperplasia, OSCAR = esophagealcancer, including cancer of the gastric-esophageal junction, GBC_CCC =gallbladder adenocarcinoma and cholangiocarcinoma, MEL = melanoma, GC =gastric cancer, UBC = urinary bladder cancer, UEC = uterine cancer.SEQ ID Peptide No. Sequence Presentation on cancer entities   1LLYPEPWSV SCLC, UEC, HNSCC   2 GLIAGVVSI HCC, UEC, HNSCC   3 KLEENGDLYLHCC, UEC, AML, HNSCC   4 KLMPGTYTL MEL, HNSCC   5 GIVAHIQEV UEC, MEL   7ILDHEVPSL HNSCC   8 SIYQFLIAV UEC, BRCA, CRC, HNSCC  10 GIAGSLKTVSCLC, MEL, AML  11 ALSPSYLTV HNSCC  12 GLLPLLHRA MEL, RCC  13 ALMAMLVYVGBC_CCC, MEL, NHL, HNSCC  14 ILAKDLFEI SCLC  15 YLDLSHNQLOSCAR, GBC_CCC, HNSCC  16 YTLDIPVLFGV HNSCC  18 ILLDLTDNRL AML, HNSCC 19 SISDNVWEV HNSCC  20 GLSQITNQL MEL, AML  21 AIQDEIRSV MEL, CRC, AML 22 FVDPNTQEKV HNSCC  23 SLFSDEFKV MEL  26 ALQEELTEL NSCLC, CRC, HNSCC 28 SLPNGKPVSV OSCAR, SCLC, HNSCC  29 YLLDPSITL HCC, SCLC, MEL, HNSCC 30 AMIEEVFEA NSCLC, HCC, UEC, GBC_CCC, NHL, HNSCC  32 VQLDSIEDLEVNSCLC, OC, UEC, MEL  33 YIKTELISV HCC  35 FLLPFSTVYL HNSCC  38 FLGPRIIGLCRC  39 HLLEGSVGV HCC, MEL  40 VLIDPQWVLTA CRC  42 LLDSVSRL CRC  43KAIEVLLTL NSCLC, AML  44 SLFETAWEA UEC, CRC, HNSCC  45 SLTEVSLPLNSCLC, CRC, HNSCC  46 SQFPLPLAV MEL, HNSCC  47 ALLERGELFVOSCAR, UBC, AML  48 QVIEDSTGV OC  49 ALNIATHVL CRC  50 ILFHGVFYA SCLC 51 LLFSRLCGA UEC  52 RLAVLFSGA HCC  53 KMVGLVVAI MEL  54 VLNPLITAVHCC, SCLC, UEC, MEL, RCC  55 SLATKIVEA AML  56 FLHDEKEGIYI AML  60ALFAPLVHL NHL  63 SQIPAQPSV AML, HNSCC  64 SLFPDSLIVNSCLC, HCC, BRCA, PC  66 KLIFSVEAV OC, UBC, BRCA  67 TLLQRLTEV NHL  69FLAVGLVDV SCLC  70 LLLGDSALYL CRC  72 FLTAINYLL UEC, RCC, AML  73YTLREVDTV AML  76 AVGPVHNSV NSCLC  77 TLIDEQDIPLV SCLC, NHL, HNSCC  85VLNDILVRA UEC  86 SVDSHFQEV NSCLC, OC  87 TIYKDFVYI OC  90 FLQDLEQRLHNSCC  91 KLFDESILI HCC, HNSCC  92 GLLFSLRSV SCLC, HNSCC  93 QVLELDVADIHNSCC  94 LLLPAVPVGA SCLC  95 GLLGSLFFLGC, OSCAR, UBC, MEL, RCC, CRC, PC, HNSCC  96 LLVSHLYLV UEC, CRC, HNSCC 98 RLFPDFFTRVAL NSCLC  100 ALLGMIIVGV HCC, AML 106 FINDSIVYLNSCLC, HCC, CRC, NHL 110 FLLGVSKEV NHL 116 TIDESGSIL MEL, AML 117KMPDVELFV NSCLC, SCLC, MEL 118 QLWQFLVTL MEL 123 VLDPALPALVNSCLC, UEC, HNSCC 126 QMFQYFITV UEC 130 SLLGGTFVGI HCC 134 GLAVPTPSVHNSCC 135 HLSTIIHEA CLL, UEC 138 ALLAKILQI HNSCC 139 FLLPTGAEAGBC_CCC, HNSCC 140 VLLEELEAL HCC, CRC, HNSCC 141 FLDKVLVAAHCC, OC, UEC, BRCA, MEL, CRC, HNSCC 143 ALLPELREV OC, SCLC, AML, HNSCC145 YLWATIQRI UEC 148 MLAGIAITV GBC_CCC, NHL 149 ILNTHITEL BRCA 150VLYDRPLKI HCC, SCLC, UEC, BRCA 151 SVLDSTAKVHCC, UEC, GBC_CCC, MEL, HNSCC 152 MMVGDLLEV UEC, HNSCC 153 FISERVEVVSCLC, UEC 154 RLLGTEFQV SCLC 155 LLNPVVEFV NSCLC, HCC, BRCA, CRC, HNSCC157 TLTSLLAQA NSCLC, CRC, HNSCC 229 AILAHLNTV UEC, HNSCC 230 KLQNIMMLLHNSCC 233 HLFDAFVSV MEL 234 LLSPHNPAL GB, HCC, SCLC, UEC, MEL, RCC 235KIIDFLSAL NSCLC, HCC, MEL, HNSCC 236 STIAILNSV MEL 237 ALAPHLDDASCLC, AML 238 GLYERPTAA GC, MCC, OSCAR, CRC, NHL 239 KMNESTRSV GB, CRC240 YMGEEKLIASV UEC, NHL 243 FMADDIFSV SCLC 244 YLLEKNRVV HNSCC 245SLLDLPLSL NSCLC, RCC, AML, HNSCC 247 ALYEGYATV AML, HNSCC 248 YLDRFLAGVSCLC, HNSCC 249 GLCERLVSL MEL, NHL, HNSCC 250 SLAPATPEV BRCA, HNSCC 252RLMEICESL UEC, MEL 253 ALAELIDNSL NSCLC, AML 254 KLQGKLPEL UEC, AML 255SLLHFTENL NSCLC, CRC 257 GLYTDPCGV HNSCC 259 ILLPRIIEAGB, GC, CLL, SCLC, UEC, BRCA, MEL, CRC, PC, HNSCC 260 ILLEKILSLGC, HCC, PCA, CLL, OC, SCLC, UEC, BRCA,GBC_CCC, MEL, PC, AML, NHL, HNSCC 261 QLQDRVYAL MEL 262 FMVDKAIYLUEC, MEL, HNSCC 263 VLLSEQGDVKL UEC, AML 266 YAIGLVMRL HCC, GBC_CCC, MEL305 KLFTSVFGV AML 306 ALLSSLNELAML, PCA, BRCA, GBC_CCC, HCC, NHL, NSCLC, OC, GB, RCC, UBC, UEC

TABLE 14A Overview of presentation of selected HLA-A*24-binding tumor-associated peptides of the present invention across entities. GB =glioblastoma, HCC = hepatocellular carcinoma, NSCLC = non-small cell lung cancer, PCA = prostate cancer, GC = gastriccancer, CRC = colorectal cell carcinoma RCC = renal cancer. SEQ IDPeptide Presentation on cancer No. Sequence entities 158 HYSQELSLLYLGB, GC, NSCLC, HCC, PCA, RCC 159 LYNKGFIYL GB, NSCLC, RCC 160 VYTLDIPVLGC, NSCLC, HCC, PCA 161 IYLVSIPEL GC, NSCLC, PCA 162 VFTRVSSFLGB, GC, NSCLC 163 DYLKGLASF GB, GC, NSCLC 164 KFSSFSLFF GC, NSCLC, PCA165 DYTTWTALL GC, NSCLC 166 YYVESGKLF GB, NSCLC, HCC 167 NYINRILKL GC168 KYQDILETI NSCLC, PCA 169 AYTLIAPNI GC, NSCLC, HCC 170 VYEDQVGKFGB, NSCLC 171 LFIPSSKLLFL NSCLC, HCC, RCC 172 TYTTVPRVAF GB, RCC 173IYSWILDHF GC, NSCLC, HCC, RCC 174 VYVGGGQIIHL GB, GC, NSCLC 175YYEVHKELF GC, NSCLC, HCC 176 EYNQWFTKL GC, NSCLC 177 VYPWLGALL GC 178IFIEVFSHF GC, NSCLC 179 MYDSYWRQF GB, NSCLC 180 IYDDSFIRPVTF NSCLC, HCC181 LYLDIINLF GC, NSCLC, PCA 182 IYQLDTASI GC, NSCLC, PCA 183 VFTSTARAFNSCLC, PCA 184 VFQNFPLLF GB, GC 185 IYKVGAPTI NSCLC 186 IFPQFLYQFGC, NSCLC 187 TYLRDQHFL GC, NSCLC, HCC 188 RYFKGLVF GB 189 WYVNGVNYFNSCLC, PCA 190 GFFIFNERF NSCLC, PCA 191 VFKASKITF GB 192 SYALLTYMI NSCLC193 RFHPTPLLL NSCLC, HCC, PCA 194 EFGSLHLEFL GB 195 TYSVSFPMFGB, GC, NSCLC, HCC, PCA 196 LYIDRPLPYL GC, NSCLC, HCC, PCA, RCC 197EYSLFPGQVVI GB, GC, NSCLC, HCC, PCA 198 LYLDKATLIGB, GC, NSCLC, HCC, PCA 199 RYAEEVGIF GC, NSCLC, HCC, PCA 200 YYGPSLFLLGC, NSCLC, RCC 201 IYATEAHVF GB, GC, NSCLC, HCC, PCA 202 VYWDSAGAAHFNSCLC 203 FYSRLLQKF GC, NSCLC 204 TYELRYFQI GB, GC, NSCLC, HCC 205VHIPEVYLI GC, NSCLC 206 EYQENFLSF NSCLC, PCA 207 AYVVFVSTL GB, GC, NSCLC208 TYTQDFNKF NSCLC, HCC 209 TYKDEGNDYF GB, NSCLC 210 IYTMIYRNLGB, GC, NSCLC 211 YYLEVGKTLI GC, NSCLC, HCC 212 YYTFHFLYF GC, NSCLC, HCC213 IFDEAEKL NSCLC, PCA 214 LYLKLWNLI NSCLC, HCC 215 YFDKVVTL NSCLC, HCC216 QYSSVFKSL GB, GC 217 FFPPTRQMGLLF GC 218 YYKSTSSAFGB, GC, NSCLC, HCC, PCA 219 EYPLVINTL GB, GC, NSCLC, HCC, RCC 220GYIDNVTLI GC, NSCLC, HCC 221 RYSTGLAGNLL GB, NSCLC, HCC, PCA 222TFSVSSHLF GB, NSCLC, HCC 223 KYIPYKYVI NSCLC, HCC 224 QYLENLEKLGB, NSCLC, HCC 225 YYVYIMNHL GB, NSCLC 226 VYRDETGELFGB, NSCLC, HCC, PCA 227 IFLDYEAGTLSF GC, RCC 228 KYTSWYVAL GB, PCA 267KYMVYPQTF GB, GC, NSCLC, HCC, PCA 268 QYLGQIQHI GB, GC, NSCLC, HCC 269YFIDSTNLKTHF GC, NSCLC, HCC 270 NYYEVHKELF GB, GC, NSCLC 271 LYHDIFSRLGC, NSCLC, HCC 272 QYLQDAYSF GB, NSCLC 273 TYIKPISKL GB, NSCLC 274AYLHSHALI NSCLC, PCA 275 EYINQGDLHEF NSCLC, PCA 276 VYGFQWRHF NSCLC 277VYQGHTALL GB, GC, NSCLC, PCA 278 RYISDQLFTNF GB, GC, NSCLC, HCC, PCA,RCC 279 TYIESASEL NSCLC, RCC 280 RYPDNLKHLYL GC, NSCLC, HCC 281PYRLIFEKF NSCLC, HCC 282 KFVDSTFYL GB, GC, NSCLC, HCC 283 TYGDAGLTYTFGB, GC, NSCLC, PCA, RCC 284 RYLNKAFHI PCA 285 HYPPVQVLFGB, GC, NSCLC, HCC, PCA 286 RYPDNLKHL NSCLC, HCC, PCA 287 LYITEPKTIGB, GC, NSCLC, HCC 288 VYVSDIQEL GC, NSCLC, HCC, PCA 289 KYPVEWAKFPCA, RCC 295 YYPGVILGF GB, NSCLC, RCC 296 TYVDSSHTIGB, GC, NSCLC, HCC, PCA 297 PFLQASPHF GC, NSCLC 298 RYLEGTSCIGB, GC, NSCLC, HCC 299 VYFVAPAKF GC, NSCLC 300 AYVLRLETLGB, GC, NSCLC, RCC 301 AYKPGALTF NSCLC, HCC 302 RYMPPAHRNF GC, NSCLC

Table 14B show the presentation on additional cancer entities forselected peptides, and thus the particular relevance of the peptides asmentioned for the diagnosis and/or treatment of the cancers asindicated.

TABLE 14B Overview of presentation of selected HLA-A*24-binding tumor-associated peptides of the presentinvention across entities. GB = glioblastoma, HCC =hepatocellular carcinoma, NSCLC = non-small cell lung cancer, PCA =prostate cancer,  GC = gastric cancer, CRC = colorectal cancer, RCC =renal cell carcinoma. SEQ ID Peptide Presentation No. Sequenceon cancer entities 162 VFTRVSSFL HCC 163 DYLKGLASF PCA 164 KFSSFSLFF PCA167 NYINRILKL NSCLC 178 IFIEVFSHF HCC 180 IYDDSFIRPVTF HCC 184 VFQNFPLLFNSCLC 206 EYQENFLSF GB 212 YYTFHFLYF PCA 216 QYSSVFKSL NSCLC, HCC 218YYKSTSSAF SCLC 227 IFLDYEAGTLSF NSCLC

TABLE 15 Presentation scores: The table lists HLA-A*02 peptides that arespecifically presented on tumors (++++), highly over-presented on tumorscompared to a panel of normal tissues (+++), highly over-presented ontumors compared to a panel of normal tissues (++) or over-presented ontumors compared to a panel of normal tissues (+). GB = glioblastoma (N =41), BRCA = breast cancer (N = 17), CRC = colorectal cancer (N = 28),RCC = renal cell carcinoma (N = 20), CLL = chronic lymphocytic leukemia(N = 17), HCC = hepatocellular carcinoma (N = 16), NSCLC = non-smalllung cancer (N = 90), SCLC = small cell lung cancer (N = 19), NHL =non-Hodgkin lymphoma (N = 18), AML = acute myeloid leukemia (N = 18), OC= ovarian cancer (N = 20), PC = pancreatic cancer (N = 16), PCA =prostate cancer and benign prostate hyperplasia (N = 39), OSCAR =esophageal cancer, including cancer of the gastric-esophageal junction(N = 17), GBC_CCC = gallbladder adenocarcinoma and cholangiocarcinoma (N= 6), MEL = melanoma (N = 7), GC = gastric cancer (N = 29), UBC =urinary bladder cancer (N = 16), UEC = uterine cancer (N = 15). Thepanel of normal tissues (N = 262) considered relevant for comparisonwith tumors consisted of: adipose tissue, adrenal gland, artery, vein,bone marrow, brain, central and peripheral nerve, eye, colon, rectum,small intestine incl. duodenum, esophagus, gallbladder, heart, kidney,liver, lung, lymph node, mononuclear white blood cells, pancreas,peritoneum, pituitary, pleura, salivary gland, skeletal muscle, skin,spleen, stomach, thymus, thyroid gland, trachea, ureter, urinarybladder. SEQ ID No. AML BRCA CLL CRC GB GBC_CCC GC HCC MEL NHL NSCLC OCOSCAR PC PCA RCC SCLC UBC UEC 1 ++++ ++++ ++++ 2 ++++ ++++ ++++ ++++++++ ++++ ++++ ++++ 3 + +++ 5 ++ + + 6 ++++ 7 ++ + + +++ 8 +++++ ++++++++ ++++ ++++ 9 ++++ ++++ ++++ ++++ 10 ++ + + 11 +++ +++ +++ +++ ++++++ 12 ++++ ++++ ++++ ++++ 13 ++++ ++++ ++++ ++++ ++++ ++++ 14 ++++ ++++++++ ++++ ++++ ++++ 15 ++++ ++++ ++++ ++++ 16 ++++ ++++ ++++ ++++ ++++17 ++++ ++++ ++++ ++++ ++++ 18 ++++ ++++ ++++ ++++ ++++ ++++ ++++ 19++++ ++++ ++++ ++++ ++++ ++++ ++++ 20 ++++ ++++ ++++ 21 ++++ ++++ ++++++++ ++++ ++++ 22 ++++ ++++ ++++ ++++ ++++ ++++ 23 ++ ++ + + + +++ 24++++ ++++ ++++ ++++ 25 ++++ ++++ 26 ++++ ++++ ++++ ++++ ++++ 27 ++++++++ ++++ ++++ ++++ 28 ++++ ++++ ++++ ++++ 29 +++ +++ +++ +++ +++ +++ 30+++ +++ +++ +++ +++ +++ 31 ++++ 32 ++++ ++++ 33 ++++ ++++ ++++ ++++ ++++34 ++++ ++++ ++++ ++++ 35 + ++ + +++ 36 ++++ ++++ 37 ++++ 38 ++++ ++++++++ 39 ++++ 40 ++++ ++++ 41 ++++ 42 ++++ ++++ ++++ 43 ++++ ++++ ++++ 44++++ ++++ ++++ ++++ 45 ++++ ++++ ++++ ++++ 46 ++++ ++++ ++++ ++++ 47++++ ++++ ++++ ++++ 48 ++++ ++++ ++++ ++++ 49 ++++ ++++ ++++ ++++ 50++++ ++++ ++++ ++++ 51 ++++ ++++ 52 ++++ ++++ ++++ ++++ 53 +++ +++ +++54 +++ +++ +++ +++ 55 +++ +++ +++ +++ 56 ++++ ++++ ++++ 57 ++++ ++++++++ ++++ 58 ++++ 59 ++++ 60 ++++ 61 ++++ ++++ ++++ 62 ++++ ++++ ++++ 63++++ ++++ ++++ 64 ++++ 65 ++++ ++++ ++++ 66 ++++ ++++ ++++ ++++ 67 ++++++ +++ +++ 68 ++++ ++++ 69 ++++ ++++ 70 ++++ ++++ 71 ++++ ++++ 72 ++++++++ 73 ++++ ++++ ++++ 74 ++++ 75 ++++ ++++ ++++ 76 ++++ ++++ 77 ++++++++ ++++ 78 ++++ ++++ ++++ 79 ++++ ++++ ++++ 80 ++++ ++++ ++++ 81 ++++++++ ++++ 82 ++++ ++++ ++++ 84 ++++ 85 ++++ ++++ 86 ++++ ++++ ++++ 87++++ ++++ ++++ 88 ++++ ++++ ++++ 89 ++++ ++++ ++++ 90 +++ + 91 + + ++ +92 + + 93 +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ 94 +++ +++ + + 95 +++++ ++ ++ + + 96 +++ +++ + +++ 97 + +++ +++ +++ 98 + 99 +++ +++ 100 +++101 ++++ 102 ++++ 103 ++++ 104 ++++ 105 ++++ 106 ++++ 107 ++++ 108 ++++109 ++++ 110 ++++ 111 ++++ ++++ ++++ 112 ++++ ++++ 113 ++++ ++++ ++++114 ++++ ++++ 115 ++++ ++++ 116 ++++ ++++ 117 + 118 ++++ 119 ++++ ++++120 ++++ ++++ 121 ++++ 122 ++++ ++++ ++++ 123 ++++ ++++ 124 ++++ ++++125 ++++ ++++ 126 ++++ ++++ 127 ++++ 128 ++++ ++++ 129 ++++ ++++ 130++++ 131 ++++ ++++ 132 ++++ ++++ 133 ++++ 134 ++++ ++++ 135 ++++ ++++136 ++++ ++++ 137 +++ +++ 138 +++ + +++ ++ +++ ++ + + + ++ + 139 +++ + +140 ++ ++ + + + +++ 141 ++ + 142 +++ + 143 + + + + 144 +++ + + +++ ++ +++++ 145 ++ + +++ + 146 +++ + 147 +++ +++ 148 +++ 149 ++++ 150 ++++ + + + 151 + ++ + + 152 +++ +++ +++ +++ + +++ 153 ++ ++ + + 154 + 155+++ ++ + 156 ++ 157 + 229 ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++++++ ++++ ++++ 230 ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++231 + 232 + ++ ++ 233 ++++ ++++ ++++ ++++ ++++ ++++ 234 ++++ ++++ ++++++++ ++++ ++++ ++++ 235 ++++ ++++ ++++ ++++ 236 ++++ ++++ ++++ ++++ ++++++++ ++++ 237 ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ 238 ++ 239 ++++++++ ++++ 240 ++ + + + 241 ++++ ++++ ++++ 242 ++++ ++++ ++++ 243 ++++244 ++++ ++++ ++++ 245 ++++ ++++ 246 ++++ ++++ ++++ 247 ++ + + +++ 248 +249 + +++ ++ + +++ 251 ++++ 252 ++++ 253 ++++ 254 ++++ ++++ 255 ++++++++ 256 ++++ ++++ 257 ++++ ++++ 258 ++++ ++++ 259 ++++ ++++ 260 ++ +++261 + + + + 262 + ++ +++ +++ + +++ 263 + + + +++ + 264 + + + 265 + +++266 +++ + 290 ++ ++ 291 ++ + + +++ ++ ++ + +++ 292 +++ +++ +++ ++ ++++++ 293 ++++ 294 ++++

TABLE 16 Presentation scores: The table lists HLA-A*24 peptides that arespecifically presented on tumors (++++), very highly over-presented ontumors compared to a panel of normal tissues (+++), highlyover-presented on tumors compared to a panel of normal tissues (++) orover-presented on tumors compared to a panel of normal tissues (+). GB =glioblastoma (N = 18), HCC = hepatocellular carcinoma (N = 15), NSCLC =non- small cell lung cancer (N = 91), PCA = prostate cancer (N = 40), GC= gastric cancer (N = 44), RCC = renal cell carcinoma (N = 2). The panelof normal tissues (N = 70) considered relevant for comparison withtumors consisted of: adrenal gland, brain, colon, rectum, heart, kidney,liver, lung, pancreas, pituitary, skin, spleen, stomach, and thymus. SEQID No. GB GC HCC NSCLC PCA RCC 158 ++++ ++++ ++++ ++++ ++++ ++++ 159++++ ++++ ++++ 160 ++++ ++++ ++++ ++++ 161 ++++ ++++ ++++ 162 ++++ ++++++++ 163 ++++ ++++ ++++ 164 ++++ ++++ ++++ 165 ++++ ++++ 166 ++++ ++++++++ 167 ++++ 168 ++++ ++++ 169 ++++ ++++ ++++ 170 ++++ ++++ 171 +++ ++++++ 172 ++++ ++++ 173 ++++ ++++ ++++ ++++ 174 ++++ ++++ ++++ 175 ++++++++ ++++ 176 ++++ ++++ 177 ++++ 178 ++++ ++++ 179 ++++ ++++ 180 ++++++++ 181 ++++ ++++ ++++ 182 ++++ ++++ ++++ 183 ++++ ++++ 184 ++++ ++++185 ++++ 186 ++++ ++++ 187 ++++ ++++ ++++ 188 ++++ 189 ++++ ++++ 190++++ ++++ 191 ++++ 192 ++++ 193 ++++ ++++ ++++ 194 ++++ 195 +++ +196 + + 197 + + 198 ++ ++ + 199 + ++ 200 + + 201 +++ + ++ 202 + 203 +204 ++ +++ + +++ 207 +++ +++ 208 + +++ 209 +++ + 210 +++ + 211 +++ 212+++ +++ +++ 213 ++ +++ 214 ++++ ++++ 215 ++++ ++++ 216 ++++ ++++ 217++++ 218 ++ + ++ 219 +++ + + ++ 222 + + 224 + + + 225 ++ 227 +++ +++ 228++ + 267 +++ +++ +++ +++ +++ 268 ++++ ++++ ++++ ++++ 269 ++++ ++++ ++++270 ++++ ++++ ++++ 271 ++++ ++++ ++++ 272 ++++ ++++ 273 ++++ ++++ 274++++ ++++ 275 ++++ ++++ 276 ++++ 277 ++ +++ + 278 + ++ 279 +++ 280 + 281+++ +++ 282 +++ +++ +++ +++ 283 + ++ +++ +++ +++ 284 ++++ 285 + + 286++ + 287 + + 288 + + + 289 +++ +++ 295 ++++ ++++ ++++ 296 297 +++ +++298 + +++ 299 ++ 300 +++ +++ +++ +++ 301 ++++ ++++ 302 ++++ ++++

Example 2

Expression Profiling of Genes Encoding the Peptides of the Invention

Over-presentation or specific presentation of a peptide on tumor cellscompared to normal cells is sufficient for its usefulness inimmunotherapy, and some peptides are tumor-specific despite their sourceprotein occurring also in normal tissues. Still, mRNA expressionprofiling adds an additional level of safety in selection of peptidetargets for immunotherapies. Especially for therapeutic options withhigh safety risks, such as affinity-matured TCRs, the ideal targetpeptide will be derived from a protein that is unique to the tumor andnot found on normal tissues.

For HLA class I-binding peptides of this invention, normal tissueexpression of all source genes was shown to be minimal based on adatabase of RNASeq data covering around 3000 normal tissue samples(Lonsdale, 2013). In addition, gene and exon expression data from tumorsvs normal tissues were analyzed to assess target coverage in varioustumor entities.

RNA Sources and Preparation

Surgically removed tissue specimens were provided as indicated above(see Example 1) after written informed consent had been obtained fromeach patient. Tumor tissue specimens were snap-frozen immediately aftersurgery and later homogenized with mortar and pestle under liquidnitrogen. Total RNA was prepared from these samples using TRI Reagent(Ambion, Darmstadt, Germany) followed by a cleanup with RNeasy (QIAGEN,Hilden, Germany); both methods were performed according to themanufacturer's protocol.

Total RNA from healthy human tissues for RNASeq experiments was obtainedfrom: Asterand (Detroit, Mich., USA & Royston, Herts, UK); BioCat GmbH(Heidelberg, Germany); BioServe (Beltsville, Md., USA); Geneticist Inc.(Glendale, Calif., USA); Istituto Nazionale Tumori “Pascale” (Naples,Italy); ProteoGenex Inc. (Culver City, Calif., USA); University HospitalHeidelberg (Heidelberg, Germany).

Total RNA from tumor tissues for RNASeq experiments was obtained from:

Asterand (Detroit, Mich., USA & Royston, Herts, UK); Bio-Options Inc.(Brea, Calif., USA); BioServe (Beltsville, Md., USA); Center for cancerimmune therapy (CCIT), Herlev Hospital (Herlev, Denmark); GeneticistInc. (Glendale, Calif., USA); Istituto Nazionale Tumori “Pascale”(Naples, Italy); Kyoto Prefectural University of Medicine (KPUM) (Kyoto,Japan); Leiden University Medical Center (LUMC) (Leiden, Netherlands);Tissue Solutions Ltd (Glasgow, UK); University Hospital Bonn (Bonn,Germany); University Hospital Geneva (Geneva, Switzerland); UniversityHospital Heidelberg (Heidelberg, Germany); Osaka City University (OCU)(Osaka, Japan); University Hospital Tübingen (Tübingen, Germany); Vald'Hebron University Hospital (Barcelona, Spain).

Quality and quantity of all RNA samples were assessed on an Agilent 2100Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 PicoLabChip Kit (Agilent).

RNAseq Experiments

Gene expression analysis of—tumor and normal tissue RNA samples wasperformed by next generation sequencing (RNAseq) by CeGaT (Tübingen,Germany). Briefly, sequencing libraries are prepared using the IIIuminaHiSeq v4 reagent kit according to the provider's protocol (IIIuminaInc., San Diego, Calif., USA), which includes RNA fragmentation, cDNAconversion and addition of sequencing adaptors. Libraries derived frommultiple samples are mixed equimolarly and sequenced on the IIIuminaHiSeq 2500 sequencer according to the manufacturer's instructions,generating 50 bp single end reads. Processed reads are mapped to thehuman genome (GRCh38) using the STAR software. Expression data areprovided on transcript level as RPKM (Reads Per Kilobase per Millionmapped reads, generated by the software Cufflinks) and on exon level(total reads, generated by the software Bedtools), based on annotationsof the ensembl sequence database (Ensembl77). Exon reads are normalizedfor exon length and alignment size to obtain RPKM values.

Exemplary expression profiles of source genes of the present inventionthat are highly over-expressed or exclusively expressed in differentcancer are shown in FIG. 2. Expression data for different entities andfurther exemplary peptides are summarized in Table 17, based on datagenerated by comparing tumor samples from the TCGA Research Network(http://cancergenome.nih.gov/.) with normal tissue samples (Lonsdale,2013). Expression scores for further exemplary genes are shown in Table18, based on in-house RNASeq analyses.

TABLE 17 Target coverage within various tumor entities, for expressionof source genes of selected peptides: A gene was consideredover-expressed if its expression level in a tumor sample was more than2-fold above the highest 75% percentile of expression levels determinedfrom high and medium risk normal tissue samples. Over-expressioncategories are indicated as “A” (>=50% of tumors above the cutoff), “B”(>=20% of tumors above the cutoff, but <50%), and “C” (>=5% of tumorsabove the cutoff, but <20%). BLCA = Bladder urothelial carcinoma (N =408), BRCA = Breast cancer (N = 1104), CHOL = Cholangiocarcinoma (N =36), COAD = Colon and rectal adenocarcinoma (N = 462), DLBC = Lymphoidneoplasm diffuse large B-cell lymphoma (N = 48), ESCA = Esophagealcancer (N = 185), GBM = Glioblastoma multiforme (N = 169), KICH = Kidneychromophobe (N = 66), KIRC = Clear cell kidney carcinoma (N = 534), KIRP= Papillary kidney carcinoma (N = 291), LAML = Acute Myeloid Leukemia (N= 173), LGG = Lower grade glioma (N = 534), LIHC = Liver hepatocellularcarcinoma (N = 374), LUAD = Lung adenocarcinoma (N = 517), LUSC = Lungsquamous cell carcinoma (N = 503), OV = Ovarian serouscystadenocarcinoma (N = 309), PAAD = Pancreatic ductal adenocarcinoma (N= 179), PRAD = Prostate adenocarcinoma (N = 498), READ = Colon andrectal adenocarcinoma (N = 167), SKCM = Skin cutaneous melanoma (N =473), STAD = Stomach adenocarcinoma (N = 415), UCEC = Uterine corpusendometrial carcinoma (N = 546). SEQ ID No. BRCA BLCA CHOL COAD DLBCESCA GBM KICH KIRC KIRP LAML LGG LIHC LUAD LUSC OV PAAD PRAD READ SKCMSTAD UCEC 1 A A 2 C C B A A A A A B A A B A B B C 3 A B B C A A A B A BA A C B A C C B C A B B 4 C C B C B 5 B C B B A C B C B C A C 6 A A 7 AA A A A A A A A A C A A A A A A A A A A A 8 B A C B A B B B C C C B C BA C B 9 B C C B C C B C C C 10 C B A C C B C 11 B C C B C B A C B C B C12 A A A A B A A A A A A A B A A A A A A A A A 13 C C 14 B B B C B C A CC C B C B B C C 15 C C C C C C B C C C 16 A B C B B A C A C C B A A C BB A C 17 A 18 C C B B C C C A C C B 19 C B B C C B C A C C C B 20 B A CA B C C C B 21 A A C A A A A A C C B A A C A A A B 22 C C B C C C 23 B BB C A C B B A C C C C B B A 24 C B C C C B B B C C C A C C 25 C C C A CA 26 B C A C 27 B B C A B C C B B B A A C A A B B B 28 C C B 29 A A A AA A A A A A B A A A A A A A A A A A 30 A B A A A B A A A A A A A A A B AA A A A A 31 C A 32 B C B B B B B B C B A A A B A 33 B B A B A A C B B CB A B B C C C B B B 34 B C A C B C A B C C B C C C B A C 35 C A 36 A 37A C 38 A 39 A C B C 40 A C 41 C 42 C B A A A B A A B A C A B B 43 B B CB A A B C A C A B B B B B 44 B C B B A C A C B C B B B 45 C C 46 C C 47B A A A A A B B C A B B B B B A B B A A A A 48 C C C C A C B C A C B B CC C A C 49 A A A A A A A C C A B C A A A A B A A A A 50 A A A A A A A AA A A A A A A A A A A A A A 51 B B B C B B C C C C C A C B B C A C 52 BB B A A A A C A A A B B A A A A A A A C 53 C B A C C B B 54 A B A B A BA A A A A A B A B B A A B A B B 55 B C 56 C A C 57 C C B C C B C 58 B AB 59 B A B C C B 60 C 61 B B B B B B B C C B A B B C B 62 C B A A A A AB A B B A B B C 63 B C A C C A B C A C A C 64 C C A C 65 A A A A A A A AA A A A A A A A A A A A A A 66 B C B A C A B C C B B B B B A C B B 67 BC A C 68 C C C C 69 B C B C C 70 B B C 71 C B C A B 72 C B A A A A 73 AA A A A A A B B B A A B A A A A B A A A A 74 C C A C C 75 B C B B C C AC C B B C B C C 76 B B C C C A C C B 77 C B B C C B C C B B C B C B B 78C A A A 79 B C C C C C C C C C C B B 80 C 81 A A A A A A A A A A A B A AA A A A A A A A 82 B B B B A B C B A C C B C C B 83 C C C C C 84 C C B BC B C C 85 C B 86 C A B B C C B C 87 C C C B B C B B C C C C 88 C B C CB B C A C B C C C B 89 A A A A A A A A A A A A A A A A A A A A A A 90 CC C C 91 B C C A C C C A C C C B C C C A C 92 C C C C B C C C C B C C CB 93 C B C B C A B C C A C C C B A C A B B B 94 B C 95 C C 96 B A A A AA C B C A A B A A A A A A B A A 97 C B B A B C C B B B C B 98 A 99 C B AB 100 C 101 C A C C 102 A 103 B 104 C A 105 B C C C C C 106 B B C B A BC C B B 107 A 108 C C C B B 109 C B B B A B B B A 110 111 B C B B B C AB B A C B B B B 112 B C C A C C B B 113 A A B A A A A C C A A B A A A BC A A A A 114 B A A A C 115 A A A A A A A A A A A A A A A A A A A A A A116 A A C A A A A C A C C B A A A A A B 117 B B A C C B A A C B C B C BC B C B 118 C A B C C C A C C 119 A B A B A B B B A A B C B A A B A B BA B B 120 C C B B A C C C A B C B A C B B A B 121 C B C B A B C C C C BC B B B B 122 C C B C C C 123 C C C C C 124 A A A A A A A B A A B B B AA A A A A A A A 125 C C 126 A A A A B A A A A B A B B A A A A A A A A A127 A A A A A A A B A A B A A A A A A A A A A A 128 A A B A A A C C A BA B A A A A B A A A A 129 C C 130 C C C C C C B C C B C C C C 131 B C BC C A C C C C C C B C 132 C C C C 133 B A 135 A B A A A A A A A A A A BA A A A A A A A B 136 C C 137 B C A B C A C C C C C C C A 138 A B C B CA C C C C A B B A C C C B A A C 139 A 140 A C 141 C A C C 142 A C B A144 B C C B A C C B C C B 145 B C 146 C B C B C B 147 C 148 A B B B A BA C B A A A C B B A B A B A B A 149 A C C C C C 150 B A B B A A B B B AB B B B C A A B B B A 151 C 152 C B A C C C B 153 A A A A B A A C C B BB B B A A B B A A A A 154 C C B A 155 A B C B C A C C C C A B B A B C CB A A C 156 B B B B B C C B C A B B A B B A A B 157 C C B C C C 229 B BB B B B B C C B B B C B B 230 B B B A A C A C B C B C B B 231 A 232 B AB A A B C C C C B B C A B B B 233 B B A A A A B C A C C B A A C A B A B234 B B C C B C B B C B 235 A A A A A A A A B A B A A A A B A A A B A236 B B A A B B A C C B B A A C B B 237 B B B A C A A C B C B B B A B BB 238 C A 239 B B A B A B A C C A B A B B B 240 B B B A A B A B C A A CB A B C B A B B B 241 A A A A A A A A A A A A A A A A A A A A A A 242 CB B C C B B 243 A C C C C 244 B C B B A A C C B B A B C B A A B 245 C AC 246 A A A A A A A C A A A A B A A A A A A A A A 247 B A A A A A C C BC C B A A B B A B A A 248 C C C C B C C C C C 249 A B C B B A C C C C AB B A B C C B A A C 250 A C C C C C C 251 B A 252 A A A A A A A C C C AB A A A A A C A A A A 253 C C B A C C C B 254 C B C C C A C C C B C B255 C B B A B B C B B C A C 256 B B A B A B A C B A B A B A B 257 C C CC C C B C C C C C C 258 A A A A A A A A A A A A A A A A A A A A A A 260C C 261 C B 262 C C C 263 C C C B A C C C C 264 A A A A A A A A A A A AA A A A A A A A A A 265 A A A A A C A A C A C A B A A A A A A A B A 266C C B C A C B C 290 A A B A A A B A C C B A A C A A A A 291 A B C A B AB A C C B A C C A B A B 292 A A A A B A A A A A A A C A A A A A A A A A293 B B C 294 C C C B C A C 158 A B C B C A C A C B A C C B A B C 159 AC C B C A C B B C A C C B A A B C B A A C 160 A B C B B A C A C C B A AC B B A C 161 C C A C C 162 A 163 A B 164 A B C C 165 C B C C A B C C CC B C C C B 166 B B B A B A B B B B A B C B A B B B A A A C 167 A A A AA A A A A A A A A A A A A A A A A A 168 C C B C A B C A B C C B 169 B BC A A A C B C C B B A A A C A C 170 A A B A B A A C A B A A B A A A A AA A A A 171 B C C B B B B C C A C C B C C A B B A 172 C A C A C 173 B AC C B 174 C B A B B C C B C B A B B 175 B B C B B A B B C C A B B B B C176 C C B C C C B C 177 B C B C 179 B B C B 180 A B B A C A A C B B A BB A A A B B A A A B 181 A A A A A A A A A A A A A A A A A A A A A A 182B B C A B A A B C C B A A A A A B B 183 C A 184 C 185 C 186 C C B A B AA C C C A 187 C A C B A B C B C C B A B C B A 188 A A 189 C C C C A C190 C A C 191 C A A C C 192 B B C B A B A B B A C C C B B B A B A 193 BA B B A B C C B C B A A C B B B B 194 C B A A C C B C C A C C 195 C C CB A C C B C B A C B B 196 A B B A B A C B A B A A B B B B B A A A A B197 B B A A A B A C A A A B A B 198 B C C B B A A B C A B C B A A C A BB C 199 A B C A A C C C C B A A B B B 200 B C A C B C A B C C B C C C BA C 201 A B B A B A B C C A C B A A C C A B A B 202 C 203 A A B A A A AC C A C C B A A C C A A A A 204 C B B A C C C C C C C C C B B 205 B A206 C 207 C C B A B C B B C C C C C 208 C B C 209 A A A A A A A B A A AA A A A A A A A A A A 210 A B B C C A B C A B A C C B B C C C C B A B211 B C A A C C B C C B 212 A C B C B A B A B C B C B B C B B C B 213 BB B B A B C B A C C B C C B 214 A A B A B B B B B B A A B A A A A A A AB A 215 C C 216 C B C C B C C C 217 B C A A B C B B A C C C C A B A A AB 218 C 219 C A A C C C B C B C C C C C A 220 C B A B A C B A A A B A B221 A A A A A A C B B A A C A A A B A A A A B 222 C B A 223 B C C A B BA C B C A 224 C C A 225 A 226 B C C 227 A A A A A A A A A A A A A A A AA A A A A A 228 C B C C C A C C C C 267 B B C C B C B B C B 268 B A A AA A A A B A C B B A A C B A A A 269 A A A A A A A A A A C A A A A A A AA A A A 270 B B C B B A B B C C A B B B B C 271 C B B A C B C C B B C BA C B C 272 B B B B A C A C A A B B A C 273 C B C B 274 C A B B 275 C CC 276 B A B A A A A A B A C B B A A C B A A B 277 B B C B B C A A A B BB B C A 278 B B C B B 279 C A A C C 280 A A A A A A A A A B A B B A A AB B A B A B 281 A B C B C A C C C C A B B A B C C B A A C 282 B A B A AA A C B A A C B A A B A A A A A 283 C C C C C B B B C C B C C B 284 B CB C A C A C C B B A B C B C 285 A A A A A A A A B A B A A A A B A A A BA 286 A A A A A A A A A B A B B A A A B B A B A B 287 B A B A A B A C AA B A B B A A B B A A B A 288 C C C B B A C C B C C C C 289 C 295 A A BC 296 A C B A 297 B C C C 298 C C B C C C 299 C B A A A B A A A A C A AB 300 B C A 301 C C C 302 B B C B A B C A A A A C C B B B A

TABLE 18 Expression scores. The table lists peptides from genes that arevery highly over-expressed in tumors compared to a panel of normaltissues (+++), highly over-expressed in tumors compared to a panel ofnormal tissues (++) or over- expressed in tumors compared to a panel ofnormal tissues (+). AML = acute myeloid leukemia (N = 11), CLL = chroniclymphocytic leukemia (N = 10), CRC = colorectal cancer (N = 20), GB =glioblastoma (N = 24), GBC_CCC = gallbladder adenocarcinoma andcholangiocarcinoma (N = 3), GC = gastric cancer (N = 11), HCC =hepatocellular carcinoma (N = 15), NHL = non-Hodgkin lymphoma (N = 10),NSCLC = non-small cell lung cancer (N = 11), OC = ovarian cancer (N =12), OSCAR = esophageal cancer, including cancer of thegastric-esophageal junction (N = 11), PC = pancreatic cancer (N = 26),PCA = prostate cancer and benign prostate hyperplasia (N = 5), RCC =renal cell carcinoma (N = 10), SCLC = small cell lung cancer (N = 10),UBC = urinary bladder cancer (N = 10), UEC = uterine cancer (N = 10).The baseline for this score was calculated from measurements of thefollowing relevant normal tissues: adipose tissue, adrenal gland,artery, blood cells, bone marrow, brain, cartilage, colon, esophagus,gallbladder, heart, kidney, liver, lung, lymph node, pancreas,pituitary, rectum, skeletal muscle, skin, small intestine, spleen,stomach, thyroid gland, trachea, urinary bladder, and vein. SEQ ID GBC_No. AML BRCA CLL CRC GB CCC GC HCC NHL NSCLC OC OSCAR PC PCA RCC SCLCUBC UEC 1 +++ 2 + + +++ ++ + +++ + + +++ + 4 ++ ++ +++ +++ + ++5 + + + + + 6 +++ 8 + + + + + + + 9 +++ + +++ ++ ++ + +++ ++ + ++ 10+++ + + + + + + ++ 11 + + + ++ + + + 12 + + + 13 + + + + 14 + +++ ++++ + + + ++ ++ + ++ + +++ + + 15 + + + + + + 16 + + + + + ++ + +++ 17 +18 ++ + ++ +++ + 19 + + 20 + + + + + + + + 21 + + + + + + +++ + + + +++ + + 22 ++ + +++ ++ + +++ +++ + + ++ 23 + + + + +++ + 24 +++ +++ ++++++ +++ + +++ 25 +++ + ++ 26 + + ++ + + + 27 + ++ +++ + + + + +++ +++ +++ + + + 28 ++ + +++ ++ 29 + + 31 +++ 32 +++ +++ +++ +++ +++ +++ ++ ++++++ +++ +++ +++ +++ +++ +++ +++ 33 ++ + +++ ++ +++ ++ + ++ + +34 + + + + + + 35 + ++ 37 + 38 + + 39 +++ + +++ + 40 + + + 41 + 42 +++++ +++ +++ +++ +++ +++ +++ +++ 43 + + ++ + + + ++ 44 + + ++ + + +++++ + + +++ + + 45 + + + ++ + + ++ + 46 + + 47 + + + 48 + +++ + + + + + + 49 + + 50 + 51 + + + 52 + + + + ++ 53 + + + + + 54 + 55 +56 + + 57 ++ + ++ +++ +++ 58 +++ + 59 +++ + +++ +++ + 60 +++61 + + + + + ++ + +++ + 62 + ++ ++ ++ + +++ + + +++ +63 + + + + + + + + + + + 64 + +++ 65 + + +++ ++ + + 67 ++ ++ ++ 68 +++ +++ ++ +++ +++ +++ ++ 69 + 70 + 71 + + 72 +++ + +++ +++ +++ 73 + + 74 +++++ 75 + + + + + + ++ + +++ + + 76 + + + + + + 78 + + 79 + + + + 80 +81 + + 82 + ++ + + + 83 ++ + +++ + + + +++ +++ 84 + 85 + +++ +++ 86 + +90 + + ++ + +++ +++ + 91 + + + + + + 93 + + + + 94 ++ + + + + ++95 + + + + 96 + 97 + + + + + + +++ + ++ ++ +++ + 98 99 ++++ + + + + + + + + ++ + 100 +++ ++ +++ + 101 +++ 102 +++ 104 +++ 105+++ +++ +++ +++ + +++ 106 +++ +++ + +++ + + +++ +++ +++ ++ +++ +++ ++107 + +++ 108 +++ +++ +++ +++ 109 +++ + +++ +++ + +++ 111 +++ + ++ + +++++ ++ + ++ 112 + + +++ + + 113 + + + + + + ++ + 114 +++ +++ 115 +116 + + + + ++ + + ++ + 117 + + + + + 118 + 119 + + 120 + + + + + + +121 + 122 ++ + +++ ++ + +++ +++ + + ++ 123 ++ + +++ + + + +++ +++127 + + + + + + + + + 128 +++ + + + + + 129 + + ++ 130 ++ + + + + + 133++ + + +++ + 134 + + + 135 136 + + + + 137 + + + + + + + + +138 + + + + + + ++ ++ + + ++ + 140 + + + 141 + + + + 142 +++ + + +144 + + + + + 145 + + + + + + +++ 146 + 147 + 148 ++ + 149 +++ + +++ ++++++ +++ +++ +++ + + ++ 150 + + ++ + + + + + + 151 + + + + + + + + + + +152 + + + + + + 153 + + + + + + 154 ++ +++ 155 + + + + + + + + + +156 + + + + + + + + + 157 ++ + +++ ++ + +++ +++ + + ++ 229 + ++ ++ + ++++ +++ +++ + +++ + + 230 + + + + + + +++ ++ ++ ++ + + +++ + ++ 231 + +232 + + + ++ + + + + + + 233 + + + + +++ + + ++ +++ + 234 ++ 235 + + + +++ + ++ + 236 + + + +++ + 237 + + + + + + + + + + 238 +239 + + + + + + + + ++ + 240 + + 241 + + + + + + + 242 ++ ++ ++ +++ +++243 ++ +++ + ++ + + 244 + + + + ++ ++ + + + + 245 + + 246 + +247 + + + + + + ++ + + + 248 + ++ + 249 + + + + + + + ++ + + + ++ + +250 ++ + + 251 +++ + 252 + ++ ++ +++ ++ + +++ +++ +++ +++ + +++ ++ +++253 + +++ + + ++ 254 + + + ++ + + + + 255 + + + ++ + ++ +++ ++ + ++ ++++ 256 + + + + ++ + + ++ + 257 + + 259 + 263 ++ + + 264 + 265 + +266 + + 290 + + + + + +++ + ++ ++ +++ + + 291 + ++ ++ ++ + + +++ +++ +++++ + +++ + + 292 +++ + 293 +++ ++ + +++ +++ + 294 158 + + + + + + + +159 + + + + + + ++ + + + + + 160 + + + + + ++ + +++ 161 + + + 163 + +164 + + + + + ++ + 165 + ++ + + + ++ + 166 + + + + + 167 + ++ + +168 + + + + 169 + + + + + ++ + + 170 + + + + + 172 +++ + + +++ ++173 + + + + 174 ++ + + 175 + + + + ++ ++ + 176 + + 177 +++ +++ +++ +++++ + + + 178 +++ +++ 179 ++ + 180 + + + + + + + + 182 + + + +++ + + 183++ + 184 + + 185 + 186 + + 187 + + +++ + ++ + + 188 +++ 189 + ++ +++ +++190 + 191 +++ 192 + + ++ + + + +++ + 193 + + + + + + + + 194 +195 + + + + + + 196 + 197 + + + 198 + + + + + ++ + + 199 +200 + + + + + + 201 + + + + + ++ + ++ ++ + ++ + + 202 +203 + + + + + + + + + + + 204 + + + ++ + 205 + +++ ++ 206 + 207 + + 208+++ +++ +++ 210 + + ++ +++ + + + + + + 211 + + + + 213 + ++ + + + 214 +++ + +++ ++ + +++ + + 215 + 216 + 217 + ++ + + ++ + + ++ +++ + + + 218++ + + + 219 ++ ++ 220 +++ +++ +++ +++ +++ +++ +++ +++ +++ 221 + + 222 +223 + + + ++ + + + 224 + 225 +++ 226 + 267 ++ 268 + + ++ ++ + ++ +270 + + + + ++ ++ + 271 ++ +++ +++ + + ++ ++ +++ +++ ++ + + ++ +++282 + + ++ + + 274 + + + 275 + 276 + + ++ + + ++ + 277 ++ + ++ + +++ +++++ + + ++ ++ +++ 278 +++ + + + 279 + + +++ + 281 + + + + + + + + +282 + + 283 + + + 284 + + + + +++ + 285 + + + + ++ + ++ + 287 + 288 ++ +++ + + + + 295 +++ 296 +++ + + + 297 ++ +++ + +++ + +++ 298 ++ + +++++ + +++ +++ + + ++ 299 + ++ +++ +++ +++ +++ +++ +++ +++ +++ 302 + + ++++++ ++ + + ++ + ++

Example 3

In Vitro Immunogenicity for MHC Class I Presented Peptides

In order to obtain information regarding the immunogenicity of theTUMAPs of the present invention, the inventors performed investigationsusing an in vitro T-cell priming assay based on repeated stimulations ofCD8+ T cells with artificial antigen presenting cells (aAPCs) loadedwith peptide/MHC complexes and anti-CD28 antibody. This way theinventors could show immunogenicity for HLA-A*0201 restricted TUMAPs ofthe invention, demonstrating that these peptides are T-cell epitopesagainst which CD8+ precursor T cells exist in humans (Table 19).

In Vitro Priming of CD8+ T Cells

In order to perform in vitro stimulations by artificial antigenpresenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28antibody, the inventors first isolated CD8+ T cells from fresh HLA-A*02leukapheresis products via positive selection using CD8 microbeads(Miltenyi Biotec, Bergisch-Gladbach, Germany) of healthy donors obtainedfrom the University clinics Mannheim, Germany, after informed consent.

PBMCs and isolated CD8+ lymphocytes were incubated in T-cell medium(TCM) until use consisting of RPMI-Glutamax (Invitrogen, Karlsruhe,Germany) supplemented with 10% heat inactivated human AB serum(PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/mlStreptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro,Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7(PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma,Nurnberg, Germany) were also added to the TCM at this step.

Generation of pMHC/anti-CD28 coated beads, T-cell stimulations andreadout was performed in a highly defined in vitro system using fourdifferent pMHC molecules per stimulation condition and 8 different pMHCmolecules per readout condition.

The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung etal., 1987) was chemically biotinylated usingSulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer(Perbio, Bonn, Germany). Beads used were 5.6 μm diameter streptavidincoated polystyrene particles (Bangs Laboratories, IIIinois, USA).

pMHC used for positive and negative control stimulations wereA*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 303) from modifiedMelan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO.304), respectively.

800.000 beads/200 μl were coated in 96-well plates in the presence of4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 wereadded subsequently in a volume of 200 μl. Stimulations were initiated in96-well plates by co-incubating 1×10⁶ CD8+ T cells with 2×10⁵ washedcoated beads in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell)for 3 days at 37° C. Half of the medium was then exchanged by fresh TCMsupplemented with 80 U/ml IL-2 and incubating was continued for 4 daysat 37° C. This stimulation cycle was performed for a total of threetimes. For the pMHC multimer readout using 8 different pMHC moleculesper condition, a two-dimensional combinatorial coding approach was usedas previously described (Andersen et al., 2012) with minor modificationsencompassing coupling to 5 different fluorochromes. Finally, multimericanalyses were performed by staining the cells with Live/dead near IR dye(Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD,Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BDLSRII SORP cytometer equipped with appropriate lasers and filters wasused. Peptide specific cells were calculated as percentage of total CD8+cells. Evaluation of multimeric analysis was done using the FlowJosoftware (Tree Star, Oreg., USA). In vitro priming of specific multimer+CD8+ lymphocytes was detected by comparing to negative controlstimulations. Immunogenicity for a given antigen was detected if atleast one evaluable in vitro stimulated well of one healthy donor wasfound to contain a specific CD8+ T-cell line after in vitro stimulation(i.e. this well contained at least 1% of specific multimer+ among CD8+T-cells and the percentage of specific multimer+ cells was at least 10×the median of the negative control stimulations).

In Vitro Immunogenicity for Different Cancer Peptides

For tested HLA class I peptides, in vitro immunogenicity could bedemonstrated by generation of peptide specific T-cell lines. Exemplaryflow cytometry results after TUMAP-specific multimer staining for 2peptides of the invention are shown in FIG. 3 together withcorresponding negative controls. Results for 10 peptides from theinvention are summarized in Table 19A. Exemplary flow cytometry resultsafter TUMAP-specific multimer staining for seven peptides of theinvention are shown in FIGS. 4 and 5 together with correspondingnegative controls. Results for 60 peptides from the invention aresummarized in Table 19B.

TABLE 19A in vitro immunogenicity of HLA class I peptidesof the invention Exemplary results of in vitroimmunogenicity experiments conducted by theapplicant for the peptides of the invention. <20% = +; 20%-49% =++; 50%-69% = +++; > = 70% = ++++ SEQ ID No. Sequence Wells 290KIVDFSYSV ++ 291 KLDETGNSL + 292 GMMTAILGV + 293 FLVDGSWSI + 295YYPGVILGF ++ 296 TYVDSSHTI + 297 PFLQASPHF ++ 298 RYLEGTSCI + 300AYVLRLETL + 301 AYKPGALTF +

TABLE 19B in vitro immunogenicity of HLA class I peptides of the invention Exemplary results of in vitro immunogenicityexperiments conducted by the applicant for thepeptides of the invention. <20% = +; 20%-49% = ++;  50%-69% = +++; > =70% = ++++ SEQ ID No Sequence Wells positive [%]   1 LLYPEPWSV ++   2GLIAGVVSI ++   4 KLMPGTYTL +   5 GIVAHIQEV +   6 ALFDSLRHV ++   7ILDHEVPSL ++  11 ALSPSYLTV ++  12 GLLPLLHRA ++++  14 ILAKDLFEI ++  18ILLDLTDNRL +  20 GLSQITNQL ++  23 SLFSDEFKV ++++  26 ALQEELTEL +  27RLMEENWNA +++  29 YLLDPSITL +  31 TITETTVEV +  35 FLLPFSTVYL +  90FLQDLEQRL +  92 GLLFSLRSV ++  96 LLVSHLYLV ++ 138 ALLAKILQI ++ 141FLDKVLVAA + 144 ALLAFFPGL +++ 149 ILNTHITEL + 150 VLYDRPLKI ++ 151SVLDSTAKV ++ 229 AILAHLNTV ++ 230 KLQNIMMLL ++++ 232 KIFPAALQLV ++ 233HLFDAFVSV ++ 235 KIIDFLSAL +++ 247 ALYEGYATV ++ 248 YLDRFLAGV ++ 249GLCERLVSL + 251 ALSVLRLAL ++ 158 HYSQELSLLYL + 159 LYNKGFIYL ++++ 160VYTLDIPVL ++ 161 IYLVSIPEL ++ 162 VFTRVSSFL ++ 163 DYLKGLASF + 165DYTTWTALL + 166 YYVESGKLF +++ 167 NYINRILKL + 168 KYQDILETI ++ 169AYTLIAPNI +++ 173 IYSWILDHF ++ 176 EYNQWFTKL +++ 196 LYIDRPLPYL ++++ 197EYSLFPGQVVI + 199 RYAEEVGIF ++ 200 YYGPSLFLL ++ 204 TYELRYFQI + 207AYVVFVSTL + 218 YYKSTSSAF + 222 TFSVSSHLF ++ 268 QYLGQIQHI + 269YFIDSTNLKTHF + 281 PYRLIFEKF +++ 285 HYPPVQVLF +

Example 4

Synthesis of Peptides

All peptides were synthesized using standard and well-established solidphase peptide synthesis using the Fmoc-strategy. Identity and purity ofeach individual peptide have been determined by mass spectrometry andanalytical RP-HPLC. The peptides were obtained as white to off-whitelyophilizates (trifluoro acetate salt) in purities of >50%. All TUMAPsare preferably administered as trifluoro-acetate salts or acetate salts,other salt-forms are also possible.

Example 5

MHC Binding Assays

Candidate peptides for T cell based therapies according to the presentinvention were further tested for their MHC binding capacity (affinity).The individual peptide-MHC complexes were produced by UV-ligandexchange, where a UV-sensitive peptide is cleaved upon UV-irradiation,and exchanged with the peptide of interest as analyzed. Only peptidecandidates that can effectively bind and stabilize the peptide-receptiveMHC molecules prevent dissociation of the MHC complexes. To determinethe yield of the exchange reaction, an ELISA was performed based on thedetection of the light chain (β2m) of stabilized MHC complexes. Theassay was performed as generally described in Rodenko et al. (Rodenko etal., 2006).

96 well MAXISorp plates (NUNC) were coated over night with 2 ug/mlstreptavidin in PBS at room temperature, washed 4× and blocked forl h at37° C. in 2% BSA containing blocking buffer. RefoldedHLA-A*02:01/MLA-001 monomers served as standards, covering the range of15-500 ng/ml. Peptide-MHC monomers of the UV-exchange reaction werediluted 100 fold in blocking buffer. Samples were incubated for 1 h at37° C., washed four times, incubated with 2 ug/ml HRP conjugatedanti-β2m for 1 h at 37° C., washed again and detected with TMB solutionthat is stopped with NH2SO4. Absorption was measured at 450 nm.Candidate peptides that show a high exchange yield (preferably higherthan 50%, most preferred higher than 75%) are generally preferred for ageneration and production of antibodies or fragments thereof, and/or Tcell receptors or fragments thereof, as they show sufficient avidity tothe MHC molecules and prevent dissociation of the MHC complexes.

TABLE 20 MHC class I binding scores. Binding of HLA-classI restricted peptides to HLA-A*02 was ranged bypeptide exchange yield: <20% = +; 20%-49% = ++; 50%-75% = +++; > = 75% =++++ SEQ ID No Sequence Peptide exchange   1 LLYPEPWSV ++++   2GLIAGVVSI +++   3 KLEENGDLYL ++++   4 KLMPGTYTL ++++   5 GIVAHIQEV +++  6 ALFDSLRHV +++   7 ILDHEVPSL +++   9 FLVDGSYSI +++  10 GIAGSLKTV +++ 11 ALSPSYLTV +++  12 GLLPLLHRA ++++  13 ALMAMLVYV ++  14 ILAKDLFEI ++++ 15 YLDLSHNQL +++  16 YTLDIPVLFGV ++++  18 ILLDLTDNRL +++  19 SISDNVWEV+++  20 GLSQITNQL +++  21 AIQDEIRSV +++  22 FVDPNTQEKV ++  23 SLFSDEFKV+++  24 TLDEKVAEL +++  25 TMDSVLVTV +++  26 ALQEELTEL +++  27 RLMEENWNA+++  28 SLPNGKPVSV +++  29 YLLDPSITL +++  30 AMIEEVFEA ++  31 TITETTVEV+++  32 VQLDSIEDLEV +++  33 YIKTELISV +++  34 FLLATEVVTV ++++  35FLLPFSTVYL +++  36 SLADTNSLAVV +++  37 ILAPFSVDL +++  38 FLGPRIIGL +++ 39 HLLEGSVGV +++  40 VLIDPQWVLTA +++  41 ALFENTPKA ++  42 LLDSVSRL + 43 KAIEVLLTL +++  44 SLFETAWEA +++  45 SLTEVSLPL +++  46 SQFPLPLAV ++ 47 ALLERGELFV +++  48 QVIEDSTGV ++  49 ALNIATHVL +++  50 ILFHGVFYA +++ 51 LLFSRLCGA ++++  53 KMVGLVVAI +++  54 VLNPLITAV +++  55 SLATKIVEA +++ 56 FLHDEKEGIYI +++  57 TVFTDHMLTV ++  58 YLLPLLPAL +++  59 KLLDPQEFTL+++  60 ALFAPLVHL +++  61 AIVKEIVNI ++  62 ALNPELVQA ++  63 SQIPAQPSV ++ 64 SLFPDSLIV ++  65 SVVPDVRSV ++  66 KLIFSVEAV +++  67 TLLQRLTEV +++ 68 SLSNRLYYL +++  69 FLAVGLVDV +++  70 LLLGDSALYL +++  71 VLHSKFWVV +++ 72 FLTAINYLL +++  73 YTLREVDTV ++  74 TLFGYSVVL +++  75 AVIKFLELL +++ 76 AVGPVHNSV ++  77 TLIDEQDIPLV +++  78 TVVTRLDEI ++  79 VTFKEYVTV ++ 80 KLYEADFVL ++  81 NALDKVLSV +++  82 FIFDEAEKL +  83 GQASYFYVA ++  84ALCPRIHEV ++++  85 VLNDILVRA +++  86 SVDSHFQEV ++  87 TIYKDFVYI ++  88AQADHLPQL ++  89 QLAPVFQRV ++  90 FLQDLEQRL +++  92 GLLFSLRSV ++++  94LLLPAVPVGA +++  95 GLLGSLFFL ++++  96 LLVSHLYLV ++  98 RLFPDFFTRVAL ++ 99 YLLQSVNQLLL ++ 100 ALLGMIIVGV + 101 ALADFMLSL +++ 102 VLLDIQEVF01+++ 103 YLVSEIFKA +++ 104 ALISWQPPRA +++ 105 ALLGTKILL +++ 106 FINDSIVYL+++ 107 LLVPTSGIYFV +++ 108 ILLKNLVTI ++ 109 SLDPSVTHL ++ 110 FLLGVSKEV+++ 111 AIVDLIHDI ++ 112 SLGKFTFDV ++++ 113 FLERGLESA ++ 114 0LIQTLHAV+++ 115 SLDPDTLPAV ++ 117 KMPDVELFV +++ 118 QLW0FLVTL +++ 119FII0GLRSVGA +++ 120 VTPVTVSAV + 121 FTIFRTISV +++ 122 GVVDPVHGV ++ 123VLDPALPALV ++ 124 KVMATIEKV ++ 125 SLADYEHFV ++++ 126 QMFQYFITV ++++ 127KLDGNELDL +++ 128 TQSPATLSV ++ 129 RLQDILWFL ++++ 130 SLLGGTFVGI +++ 131VTSNSGILGV +++ 132 ILGEVLAQL +++ 133 ALLPRLHQL ++++ 134 GLAVPTPSV +++135 HLSTIIHEA +++ 136 FLFGGVLMTL ++ 138 ALLAKILQI +++ 139 FLLPTGAEA ++141 FLDKVLVAA +++ 142 ILVEGISTV +++ 143 ALLPELREV +++ 144 ALLAFFPGL +++145 YLWATIQRI +++ 146 ALHFSEDEI ++ 147 YLMDDTVEI ++++ 148 MLAGIAITV +++149 ILNTHITEL +++ 150 VLYDRPLKI +++ 151 SVLDSTAKV ++ 152 MMVGDLLEV +++153 FISERVEVV ++++ 154 RLLGTEFQV +++ 155 LLNPVVEFV +++ 156 ILGDLSHLL +++157 TLTSLLAQA +++ 229 AILAHLNTV ++++ 230 KLQNIMMLL ++ 231 MLDKYSHYL +++232 KIFPAALQLV +++ 233 HLFDAFVSV +++ 234 LLSPHNPAL +++ 235 KIIDFLSAL +++236 STIAILNSV +++ 237 ALAPHLDDA +++ 238 GLYERPTAA ++ 239 KMNESTRSV ++240 YMGEEKLIASV +++ 241 KTIQQLETV ++ 242 WLYGEDHQI ++ 243 FMADDIFSV +++244 YLLEKNRVV ++ 245 SLLDLPLSL ++++ 246 TVSDVLNSV +++ 247 ALYEGYATV +++248 YLDRFLAGV ++++ 249 GLCERLVSL ++++ 250 SLAPATPEV +++ 251 ALSVLRLAL+++ 252 RLMEICESL +++ 253 ALAELIDNSL +++ 254 KLQGKLPEL ++ 255 SLLHFTENL+++ 256 SLGEEQFSV ++ 257 GLYTDPCGV ++++ 258 LLSERFINV ++++ 259 ILLPRIIEA+++ 260 ILLEKILSL +++ 261 QLQDRVYAL +++ 262 FMVDKAIYL +++ 263VLLSEQGDVKL ++ 264 KLFPQETLFL +++ 265 NTCPYVHNI +++ 266 YAIGLVMRL +++

TABLE 21 MHC class I binding scores. Binding of HLA-classI restricted peptides to HLA-A*24 was ranged bypeptide exchange yield: <20% = +; 20%-49% = ++; 50%-75% = +++; > = 75% =++++ SEQ ID No Sequence Peptide exchange 172 TYTTVPRVAF ++++ 177VYPWLGALL ++++ 178 IFIEVFSHF +++ 179 MYDSYWRQF ++++ 180 IYDDSFIRPVTF +++181 LYLDIINLF ++++ 182 IYQLDTASI +++ 183 VFTSTARAF +++ 184 VFQNFPLLF++++ 185 IYKVGAPTI +++ 186 IFPQFLYQF ++++ 187 TYLRDQHFL ++++ 188RYFKGLVF +++ 189 WYVNGVNYF ++ 190 GFFIFNERF +++ 191 VFKASKITF +++ 192SYALLTYMI ++++ 193 RFHPTPLLL ++++ 194 EFGSLHLEFL + 198 LYLDKATLI +++ 203FYSRLLQKF +++ 205 VHIPEVYLI +++ 206 EYQENFLSF +++ 208 TYTQDFNKF +++ 210IYTMIYRNL ++++ 211 YYLEVGKTLI ++++ 214 LYLKLWNLI +++ 215 YFDKVVTL ++ 216QYSSVFKSL ++++ 217 FFPPTRQMGLLF ++++ 219 EYPLVINTL +++ 220 GYIDNVTLI++++ 221 RYSTGLAGNLL +++ 223 KYIPYKYVI +++ 224 QYLENLEKL +++ 225YYVYIMNHL +++ 226 VYRDETGELF +++ 227 IFLDYEAGTLSF ++++ 228 KYTSWYVAL++++ 267 KYMVYPQTF ++++ 271 LYHDIFSRL ++++ 272 QYLQDAYSF ++++ 273TYIKPISKL +++ 274 AYLHSHALI ++++ 275 EYINQGDLHEF +++ 276 VYGFQWRHF ++++278 RYISDQLFTNF ++++ 279 TYIESASEL +++ 280 RYPDNLKHLYL ++++ 282KFVDSTFYL ++++ 283 TYGDAGLTYTF +++ 284 RYLNKAFHI +++ 286 RYPDNLKHL ++288 VYVSDIQEL +++ 289 KYPVEWAKF ++++ 158 HYSQELSLLYL ++++ 159 LYNKGFIYL++++ 160 VYTLDIPVL ++++ 161 IYLVSIPEL ++++ 162 VFTRVSSFL +++ 163DYLKGLASF ++++ 164 KFSSFSLFF +++ 165 DYTTWTALL ++++ 166 YYVESGKLF ++++167 NYINRILKL ++++ 168 KYQDILETI ++++ 169 AYTLIAPNI +++ 170 VYEDQVGKF ++171 LFIPSSKLLFL +++ 173 IYSWILDHF ++++ 174 VYVGGGQIIHL +++ 175 YYEVHKELF++++ 176 EYNQWFTKL +++ 195 TYSVSFPMF ++++ 196 LYIDRPLPYL +++ 197EYSLFPGQVVI +++ 199 RYAEEVGIF +++ 200 YYGPSLFLL +++ 201 IYATEAHVF +++202 VYWDSAGAAHF ++++ 204 TYELRYFQI ++++ 207 AYVVFVSTL ++ 209 TYKDEGNDYF+++ 218 YYKSTSSAF +++ 222 TFSVSSHLF +++ 268 QYLGQIQHI +++ 269YFIDSTNLKTHF +++ 270 NYYEVHKELF +++ 277 VYQGHTALL ++++ 281 PYRLIFEKF ++285 HYPPVQVLF +++ 287 LYITEPKTI +++

Example 6

Absolute Quantitation of Tumor Associated Peptides Presented on the CellSurface

The generation of binders, such as antibodies and/or TCRs, is alaborious process, which may be conducted only for a number of selectedtargets. In the case of tumor-associated and—specific peptides,selection criteria include but are not restricted to exclusiveness ofpresentation and the density of peptide presented on the cell surface.In addition to the isolation and relative quantitation of peptides asdescribed in Error! Reference source not found, the inventors didanalyze absolute peptide copies per cell as described. The quantitationof TUMAP copies per cell in solid tumor samples requires the absolutequantitation of the isolated TUMAP, the efficiency of TUMAP isolation,and the cell count of the tissue sample analyzed.

Peptide Quantitation by nanoLC-MS/MS

For an accurate quantitation of peptides by mass spectrometry, acalibration curve was generated for each peptide using the internalstandard method. The internal standard is a double-isotope-labelledvariant of each peptide, i.e. two isotope-labelled amino acids wereincluded in TUMAP synthesis. It differs from the tumor-associatedpeptide only in its mass but shows no difference in otherphysicochemical properties (Anderson et al., 2012). The internalstandard was spiked to each MS sample and all MS signals were normalizedto the MS signal of the internal standard to level out potentialtechnical variances between MS experiments. The calibration curves wereprepared in at least three different matrices, i.e. HLA peptide eluatesfrom natural samples similar to the routine MS samples, and eachpreparation was measured in duplicate MS runs. For evaluation, MSsignals were normalized to the signal of the internal standard and acalibration curve was calculated by logistic regression. For thequantitation of tumor-associated peptides from tissue samples, therespective samples were also spiked with the internal standard, the MSsignals were normalized to the internal standard and quantified usingthe peptide calibration curve.

Efficiency of Peptide/MHC Isolation

As for any protein purification process, the isolation of proteins fromtissue samples is associated with a certain loss of the protein ofinterest. To determine the efficiency of TUMAP isolation, peptide/MHCcomplexes were generated for all TUMAPs selected for absolutequantitation. To be able to discriminate the spiked from the naturalpeptide/MHC complexes, single-isotope-labelled versions of the TUMAPswere used, i.e. one isotope-labelled amino acid was included in TUMAPsynthesis. These complexes were spiked into the freshly prepared tissuelysates, i.e. at the earliest possible point of the TUMAP isolationprocedure, and then captured like the natural peptide/MHC complexes inthe following affinity purification. Measuring the recovery of thesingle-labelled TUMAPs therefore allows conclusions regarding theefficiency of isolation of individual natural TUMAPs.

The efficiency of isolation was analyzed in a low number of samples andwas comparable among these tissue samples. In contrast, the isolationefficiency differs between individual peptides. This suggests that theisolation efficiency, although determined in only a limited number oftissue samples, may be extrapolated to any other tissue preparation.However, it is necessary to analyze each TUMAP individually as theisolation efficiency may not be extrapolated from one peptide to others.

Determination of the Cell Count in Solid, Frozen Tissue

In order to determine the cell count of the tissue samples subjected toabsolute peptide quantitation, the inventors applied DNA contentanalysis. This method is applicable to a wide range of samples ofdifferent origin and, most importantly, frozen samples (Alcoser et al.,2011; Forsey and Chaudhuri, 2009; Silva et al., 2013). During thepeptide isolation protocol, a tissue sample is processed to a homogenouslysate, from which a small lysate aliquot is taken. The aliquot isdivided in three parts, from which DNA is isolated (QiaAmp DNA Mini Kit,Qiagen, Hilden, Germany). The total DNA content from each DNA isolationis quantified using a fluorescence-based DNA quantitation assay (QubitdsDNA HS Assay Kit, Life Technologies, Darmstadt, Germany) in at leasttwo replicates.

In order to calculate the cell number, a DNA standard curve fromaliquots of single healthy blood cells, with a range of defined cellnumbers, has been generated. The standard curve is used to calculate thetotal cell content from the total DNA content from each DNA isolation.The mean total cell count of the tissue sample used for peptideisolation is extrapolated considering the known volume of the lysatealiquots and the total lysate volume.

Peptide Copies Per Cell

With data of the aforementioned experiments, the inventors calculatedthe number of TUMAP copies per cell by dividing the total peptide amountby the total cell count of the sample, followed by division throughisolation efficiency. Copy cell number for selected peptides are shownin Table 22.

TABLE 22 Absolute copy numbers. The table lists theresults of absolute peptide quantitation intumor samples. The median number of copies percell are indicated for each peptide: <100 = +; > = 100 = ++; > =1,000 +++; > = 10,000 = ++++. The number of samples, in which evaluable,high quality MS data is available is indicated. SEQ ID Copies perNumber of No. Peptide Code cell (median) samples   2 MET-007 + 15  24MAGEC2-001 + 16  32 PRAME-006 ++ 17  39 ABCC11-001 + 14 251 SPINK2-001++ 16

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The invention claimed is:
 1. A method for the treatment of a patient whohas cancer, comprising administering to the patient a population ofactivated T lymphocyte that selectively recognizes cancer cell, whichpresents a peptide consisting of the amino acid sequence of HLLEGSVGV(SEQ ID NO: 39), wherein the peptide is in a complex with an MHCmolecule, wherein said cancer is selected from the group consisting ofglioblastoma, breast cancer, colorectal cancer, renal cell carcinoma,chronic lymphocytic leukemia, hepatocellular carcinoma, non-small celland small cell lung cancer, Non-Hodgkin lymphoma, acute myeloidleukemia, ovarian cancer, pancreatic cancer, prostate cancer, esophagealcancer including cancer of the gastric-esophageal junction, gallbladdercancer and cholangiocarcinoma, melanoma, gastric cancer, testis cancer,urinary bladder cancer, head and neck squamous cell carcinoma, anduterine cancer.
 2. The method of claim 1, wherein the T cells areautologous to the patient.
 3. The method of claim 1, wherein the T cellsare obtained from a healthy donor.
 4. The method of claim 1, wherein theT cells are obtained from tumor infiltrating lymphocytes or peripheralblood mononuclear cells.
 5. The method of claim 1, wherein the activatedT cells are expanded in vitro.
 6. The method of claim 1, wherein the MHCmolecule is a class I MHC molecule.
 7. The method of claim 1, whereinthe antigen presenting cell is infected with a recombinant virusexpressing the peptide.
 8. The method of claim 7, wherein the antigenpresenting cell is a dendritic cell or a macrophage.
 9. The method ofclaim 5, wherein the expansion is in the presence of an anti-CD28antibody and IL-12.
 10. The method of claim 1, wherein the population ofactivated T cells comprises CD8-positive cells.
 11. The method of claim1, wherein the contacting is in vitro.
 12. The method of claim 1,wherein the population of activated T cells are administered in the formof a composition.
 13. The method of claim 12, wherein the compositioncomprises an adjuvant.
 14. The method of claim 13, wherein the adjuvantis selected from the group consisting of imiquimod, resiguimod, GM-CSF,cyclophosphamide, Sunitinib, bevacizumab, interferon-alpha, CpGoligonucleotides and derivatives, poly-(I:C) and derivatives, RNA,sildenafil, and particulate formations with poly(lactid co-glycolid)(PLG) and virosomes.
 15. The method of claim 1, wherein the immuneresponse is capable of killing targeted cancer cells.
 16. The method ofclaim 1, wherein the immune response is capable of killing the cancercells.
 17. The method of claim 15, wherein the immune response comprisesa cytotoxic T cell response.
 18. The method of claim 1, wherein theimmune response comprises a cytotoxic T cell response.
 19. The method ofclaim 1, wherein the cancer is breast cancer.
 20. The method of claim 1,wherein the cancer is prostate cancer.